Fig. 5. Intestinal structure is not affected by diet or gene deletion. (A and B) Mean villus height (A) and crypt depth (B) of intestinal tissues from wild-type mice maintained on low- (L) or high- (H) carbohydrate diets or on the low-carbohydrate diet supplemented with drinking water containing 20 mM saccharin (sac), 1 mM aspartame (asp), or 10 mM acesulfame K (ace-K). There is no statistically significant change observed in the crypt depth or villus height in the intestines of mice on these various diets. (C), Western blots of villin and b-actin in wild-type (WT) and Ga_gust and T1R3 knockout mice on both low- (L) and high- (H) carbohydrate diets show no significant differences in levels of these proteins between wild-type and knockout mice and no change in the level of either protein as a function of dietary carbohydrate. Villin protein gives a good indication of brush-border membrane recovery and purity, both of which are unaffected by diet or gene deletion. Note that the weaker signal for low-abundance villin vs. high-abundance b-actin is due to adjusting the exposure time so that both signals are measured within the linear range for densitometric analysis. Statistical significance determined by ANOVA, data are expressed as means ±SD.

Fig. 6. In situ hybridization controls. In situ hybridization histochemistry with complementary sense probes to T1R2, T1R3, Ga_gust, and SGLT1 did not nonspecifically hybridize with targets within the mouse small intestine.

Fig. 7. Immunohistochemistry controls. Omission of the primary antibodies for gustducin and T1R3 with mouse (FITC Mouse and Cy3 Mouse, respectively) and human (FITC Human and Cy3 Human, respectively) tissues showed no nonspecific immunoreactivity with targets within the mouse small intestine.

Fig. 8. Detection of taste signaling elements in enteroendocrine cell lines. Real-time qPCR indicates expression of the T1R taste receptor subunits and Ga_gust in the mouse enteroendocrine cell lines STC-1 and GLUTag. Data are expressed as means ± SD. All data were generated in triplicate, with n = 4 samples in each group.

Fig. 9. Gurmarin inhibits calcium mobilization in sucralose-stimulated GLUTag cells. Intracellular free Ca²⁺ in GLUTag cells transfected, to maximally enhance detection of calcium mobilization, with Ga16/gust44, YC3.60, and REEP-EI was monitored continuously by fluorescence confocal microscopy. The addition of sucralose (20 mM final concentration, arrow) led to an increase in intracellular free Ca²⁺ [(Upper) solid line; (Lower) top row of cells). Preincubation (15 min) of the transfected GLUTag cells with gurmarin (1 mg/ml) blocked the Ca²⁺ response to sucralose [(Upper) dashed line; (Lower) middle row of cells]. Control GLUTag cells transfected with YC3.60 and REEP-EI, but lacking Ga16/gust44, gave no Ca²⁺ response above baseline to sucralose [(Upper) dotted line; (Lower) bottom row of cells]. Confocal images shown depict intracellular calcium concentrations at time points 0, 40 s, and 100 s (sucralose was added at t = 10 s). (Scale bars, 10 mm.) Traces are from representative single cells.

Fig. 10. Gurmarin inhibits mouse T1R2+T1R3 sweet taste receptor activity. HEK 293 cells were transfected with plasmids encoding mouse T1R2, mouse T1R3, and Ga16/gust44 loaded with the fluorescent calcium indicator dye Fluo-4 and then exposed to sweeteners with or without the mouse sweet taste inhibitor gurmarin. Fluorescence responses were recorded using a FlexStationII fluorimeter. (A) Fluo-4 Fluorescence, in arbitrary units (a.u.), measures calcium mobilization in response to sweet taste receptor activation by the addition of 10 mM sucralose (Suc) (open circles). Preincubation (15 min) with gurmarin (1 mg) abolishes the response (filled circles). (B) DeltaF/F measures the peak amplitude of calcium mobilization in response to addition of sweeteners: 10 mM sucralose (Suc), 10 mM saccharin (Sac) and 10 mM acesulfame-K (AceK) and 20 mM cyclamate (Cyc). The expressed mouse T1R2+T1R3 receptor does not respond to the human-specific sweetener cyclamate [see Jiang P, Cui M, Zhao B, Liu Z, Snyder LA, Benard LMJ, Osman R, Margolskee RF, Max M (2005) J Biol Chem 280:34296-34305]. Preincubation (15 min) with gurmarin (1 mg) abolishes the responses to sucralose, saccharin, and acesulfame-K.