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Detection of Sweet and Umami Taste in the Absence of Taste Receptor T1r3

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Science  08 Aug 2003:
Vol. 301, Issue 5634, pp. 850-853
DOI: 10.1126/science.1087155

Abstract

The tastes of sugars (sweet) and glutamate (umami) are thought to be detected by T1r receptors expressed in taste cells. Molecular genetics and heterologous expression implicate T1r2 plus T1r3 as a sweet-responsive receptor,and T1r1 plus T1r3,as well as a truncated form of the type 4 metabotropic glutamate receptor (taste-mGluR4),as umami-responsive receptors. Here,we show that mice lacking T1r3 showed no preference for artificial sweeteners and had diminished but not abolished behavioral and nerve responses to sugars and umami compounds. These results indicate that T1r3-independent sweet- and umami-responsive receptors and/or pathways exist in taste cells.

The sac gene in mice is the major genetic determinant regulating behavioral and nerve responses to artificial sweeteners, such as saccharin, and to several sugars (16). Recently, the taste receptor T1r3 was identified as the sac gene product (712). Heterologously expressed T1r3 appears not to function on its own. However, in combination with T1r2 it responds to many sweet compounds, and in combination with T1r1 it responds to glutamate and other umami compounds (11, 13, 14). To determine the role of T1r3 in vivo, we produced knockout (KO) mice lacking the entire T1r3 coding region by homologous recombination in C57BL6 (B6) embryonic stem (ES) cells and then injected the targeted stem cells into blastocysts (Fig. 1, A and B). T1r3 protein was absent in T1r3 KO mice, as demonstrated by indirect immunofluorescence (Fig. 1, C and D). The T1r3 KO mice were healthy and fertile with no obvious anatomical or behavioral abnormalities. The gross anatomy of the taste tissue and number of taste buds appeared normal in the T1r3 KO mice (Fig. 1F). Knocking out the T1r3 gene did not alter expression of T1r1 (15) or T1r2 (Fig. 1, E and F).

Fig. 1.

Generation of T1r3 knockout mice. (A) (Top) A map of the murine T1r3 locus showing the six exons (filled boxes), and intervening and flanking sequences. The polymerase chain reaction (PCR) primers that were used to detect the wild-type T1r3 allele are indicated by the numbered short lines below the map. (Middle) A map of the targeting vector showing PGKneo (neo) and PGKTK (TK) genes (open boxes) and the long and short arms of the targeting vector. PGKneo was flanked with LoxP sites for removal of neo by Cre recombinase. The arrow indicates the direction of transcription of neo. The targeting vector was designed to remove the entire T1r3 coding region. (Bottom) A diagram of the T1r3 targeted allele. The primers that were used to screen the G418-resistant ES colonies are indicated by numbered short lines below the map. (B) The offspring from the cross of two T1r3 heterozygotes (+/–) were genotyped by PCR amplification of mouse tail DNA. (Top) PCR amplification of T1r3 exons 1 and 2 with primers 1 and 2 [indicated in the top line of (A)]. (Bottom) PCR amplification of upstream T1r3 sequences and neo with primers 3 and 4 [indicated in bottom line of (A)], indicative of successful targeting. (C and D) Photomicrographs of frozen sections of taste bud–containing circumvallate papillae from wild-type (C) and T1r3 KO (D) mice, stained with T1r3-specific antibodies (red), show that T1r3 protein is absent in the T1r3 KO mice. (E and F) Photomicrographs of frozen sections of taste bud–containing circumvallate papillae from wild-type (E) and T1r3 KO (F) mice, labeled by in situ hybridization with a T1r2 probe (blue), show that expression of T1r2 is not affected by the absence of T1r3.

Behavioral tests (16) were conducted to examine the responses of T1r3 KO mice to tastants representing five taste qualities (sweet, bitter, salty, sour, and umami). In two-bottle preference tests, the T1r3 KO mice displayed indifference to sucrose and three artificial sweeteners (sucralose, acesulfame K, and SC45647) at concentrations that elicited maximal preference in B6 wild-type littermate controls (Fig. 2). At concentrations that were 5 to 10 times as high as those needed to elicit a strong preference in B6 wild-type mice, the T1r3 KO mice preferred sucrose, but avoided all three artificial sweeteners. The response of the T1r3 KO mice to glucose was slightly reduced as compared with that of the B6 wild-type controls (Fig. 2) but was not significant at the P < 0.05 level (P = 0.074; Table 1). There was no difference between the responses of T1r3 KO mice and B6 wild-type mice to maltose (fig. S1). Compared with B6 wild-type mice, the preference of T1r3 KO mice for monosodium glutamate (MSG) concentrations between 30 and 300 mM was reduced but not abolished (Fig. 2). T1r3 KO mice and B6 wild-type mice showed similar responses to quinine sulfate, HCl, and NaCl (fig. S1), indicating that detection of bitter, sour, and salty compounds, respectively, does not involve T1r3.

Fig. 2.

Mean preference ratios from 48-hour two-bottle preference tests (tastant versus water) comparing T1r3–/– (T1r3 KO mice, open circles), T1r3+/+ and T1r3+/– littermates [C57BL6 (B6) wild-type mice, red circles], and 129T2/SvemsJ (129 wild-type mice, green triangles) mice. The mice were given two bottles, one containing water and the other a tastant solution. The ratio of tastant to total liquid consumed after 48 hours was measured and compared between groups. T1r3 KO mice displayed markedly diminished behavioral responses to all three artificial sweeteners and sucrose, and moderately diminished behavioral responses to MSG (Table 1 lists F and P values). For responses to glucose, the difference between T1r3 KO mice and B6 wild-type mice was borderline (P = 0.07). For each group, n = 10. Error bars are the SEM.

Table 1.

Summary of statistical analysis of behavioral and electrophysiological data. The general linear model for repeated measurements analysis was used to compare the responses of T1r3 KO, B6 wild-type, and 129 wild-type mice, with tastant concentrations as dependent variables and genotype as a fixed factor. For each compound, the responses from all concentrations were analyzed together, with the response from each concentration treated as a single value for the multiple measurement analysis. For behavioral tests, the F value is given for comparison of all genotypes. When a significant difference was found, the Tukey's test was used to determine which of the means differed. P values from the Tukey's test are given for T1r3 KO versus B6 mice and for T1r3 KO versus 129 mice. When this analysis gave a borderline P value (0.05 < P < 0.06), each concentration was analyzed individually by the general linear model univariate analysis. For CT and GL nerve responses to glucose, maltose, and sucralose, the general linear model univariate analysis was used because only one concentration was tested for each of these tastants. ND, not done.

Tastant Two-bottle preference CT nerve GL nerve
FP(KO vs B6)P(KO vs 129)FPFP
Sucrose 21.144 <0.001 0.109 37.359 <0.001 4.894 0.057View inline
Glucose 2.947 0.074 0.889 3.080 0.117 ND ND
Maltose 3.361 0.500 0.316 9.678 0.014 ND ND
Sucralose 52.316 <0.001 0.003 17.360 0.003 ND ND
AceK 38.434 <0.001 0.055View inline 26.341 0.001 14.692 0.006
SC45647 430.076 <0.001 <0.001 19.705 0.004 9.79 0.02
MSG 9.607 0.006 ND 4.815 0.056View inline 0.216 0.654
NaCl 0.564 0.464 ND 0.558 0.476 0.006 0.940
Quinine 0.933 0.349 ND 1.696 0.234 0.417 0.542
HCl 1.207 0.289 ND 0.440 0.525 0.227 0.650
  • View inline* P = 0.036 for 300 mM, P = 0.003 for 500 mM and 1 M.

  • View inline P = 0.017 for 30 mM, P < 0.001 for 100 mM.

  • View inline P = 0.027 for 1000 mM.

  • B6 mice are classified as “tasters” because of their strong preference for sucrose, saccharin, and other sweet compounds (1, 2). In contrast, 129T2/SvemsJ (129) “nontaster” mice require higher concentrations of sweet compounds to elicit preference (6). To determine if T1r3 in 129 mice is completely non-functional (null) or partially functional (a hypomorph), we compared the behavioral responses to sweet compounds of B6 (taster), 129 (nontaster), and T1r3 KO (null) mice. Preference responses to all three artificial sweeteners (sucralose, acesulfame K, and SC45647) were strongest in B6 mice, weakest in T1r3 KO mice, and intermediate in 129 mice; the mean response of T1r3 KO mice to sucrose was weaker than that of 129 mice (Fig. 2). These results suggest that T1r3 is not a null allele in 129 mice, but has reduced functionality for these sweet compounds. The 129 mice did not differ from T1r3 KO mice in their responses to glucose and maltose (P = 0.889 and 0.316, respectively, Table 1; Fig. 2; Fig. S1).

    To examine the contributions of the gustatory nerves to the behavioral responses, we recorded from the chorda tympani (CT) branch of the facial nerve and the glossopharyngeal (GL) nerve of T1r3 KO and B6 wild-type mice (16). Whole-nerve recordings from the CT nerve of T1r3 KO mice showed near-zero responses to all four artificial sweeteners tested (acesulfame K, SC45647, saccharin, and sucralose) (Fig. 3, A and C; fig. S2). Compared with the CT responses of B6 wild-type mice, the CT responses of the T1r3 KO mice to sweet-tasting amino acids, sugar alcohols, and sugars ranged from near-zero (D-tryptophan), to moderately or markedly diminished (sucrose, fructose, maltose, and sorbitol), to not significantly diminished (glucose) (Fig. 3C). Compared with the CT responses of B6 wild-type mice, the CT responses of T1r3 KO mice to MSG were diminished only at the highest concentration tested (Fig. 3A). Inosine monophosphate (IMP) is a known potentiator of umami responses, enhancing behavioral and CT nerve responses to MSG by about one order of magnitude (17). Compared with the B6 wild-type mice, the T1r3 KO mice displayed markedly reduced CT responses to MSG+IMP (umami) (Fig. 3A). Indeed, the CT responses of T1r3 KO mice to MSG+IMP were indistinguishable from those to MSG alone, as if they had lost the potentiating effect of IMP. T1r3 KO and B6 control mice showed similar responses to quinine sulfate, HCl, and NaCl (fig. S2), consistent with the behavioral data.

    Fig. 3.

    Whole-nerve recordings from taste nerves of B6 wild-type and T1r3 KO mice stimulated by lingual application of taste stimuli. (A) Integrated CT responses of wild-type mice (red circles) and T1r3 KO mice (open circles) in response to lingual application of a concentration series of tastants, normalized to the response to 100 mM NH4Cl (NH4Cl response = 1.0). Compared with the responses of B6 wild-type mice, CT responses of T1r3 KO mice were markedly diminished to acesulfame K, SC45647, sucrose, and MSG+IMP, and slightly diminished to MSG (Table 1 lists F and P values). In the MSG+IMP graph, the dotted curve shows the response of the B6 wild-type mice to MSG alone, and the dashed curve shows the response of T1R3 KO mice to MSG alone. (B) Integrated glossopharyngeal (GL) responses of wild-type (red circles) and T1r3 KO (open circles) mice in response to lingual application of tastants, normalized to the response to 100 mM NH4Cl (NH4Cl response = 1.0). In general, responses to sweet compounds were lower in the GL than in the CT. Significant differences were noted between T1r3 KO mice and B6 wild-type mice in responses to acesulfame K, SC45647, sucrose, and MSG+IMP but not in response to MSG alone (Table 1 lists F and P values). For MSG+IMP, P < 0.001 and F = 40.848. (C) Integrated CT responses of wild-type (red bars) and T1r3 KO (open bars) mice in response to lingual application of single concentrations of sweet tastants, normalized to the response to 100 mM NH4Cl (NH4Cl response = 1.0). In addition to the T1r3 KO mice versus wild-type mice differences noted in (A), significant differences were observed with maltose (Table 1 lists F and P values), fructose (F = 39.8, P < 0.001), sorbitol (F = 9.9, P = 0.014), and d-tryptophan (F = 19.8, P = 0.002). For (A) and (B), all concentrations are in millimolar. For (C), the following single concentrations were tested: 500 mM glucose, 500 mM maltose, 500 mM fructose, 1 M sorbitol, 300 mM sucrose, 20 mM saccharin, 8 mM SC45647, 25 mM sucralose, and 30 mM d-tryptophan. Error bars are the SEM. For each group and each nerve, n = 5. For details and results of the statistical analysis, see Table 1.

    T1r3 is expressed in both anterior and posterior tongue regions (712), so it was of interest to know what effect its absence from posterior taste buds might have on GL nerve responses. In rodents, the GL nerve is relatively more responsive to bitter compounds, whereas the CT nerve is relatively more responsive to sweet compounds (1820). As expected, the GL nerve responses of B6 wild-type mice to the sweet compounds SC45647 and sucrose were much lower than were the CT responses to these compounds (Fig. 3, A and B; fig. S2). Compared with the CT nerve responses, the GL nerve responses of B6 wild-type mice to acesulfame K were lower at concentrations less than 100 mM but higher at 300 mM. This may explain the averseness to higher concentrations of acesulfame K evident in the behavioral assays (Fig. 2). Compared with B6 wild-type mice, the T1r3 KO mice showed reduced GL responses to the three sweet compounds tested (sucrose, SC45647, and acesulfame K); for sucrose and SC45647, this was only evident at the highest concentration tested, whereas for acesulfame K, this was evident at concentrations of 30 to 300 mM (Fig. 3B and fig. S2). In contrast to the CT results, no differences between B6 wild-type mice and T1r3 KO mice were noted in the GL responses to either MSG alone or MSG+IMP (Fig. 3B), suggesting that T1r3 does not underlie the umami responses mediated by the GL nerve. GL responses to quinine sulfate, HCl, and NaCl were unchanged in T1r3 KO mice as compared with the B6 wild-type mice (fig. S2), consistent with the behavioral and CT data.

    The behavioral data from T1r3 KO mice show that T1r3 is the primary or only taste receptor determining preference for the artificial sweeteners. T1r3 KO mice detect and avoid high concentrations of these compounds, implying the existence of other artificial sweetener–responsive receptors (such as T2rs) that may mediate the well-known bitter aftertaste of these sweeteners (21). T1r3 KO mice displayed significant behavioral and/or gustatory nerve responses to all of the sugars tested, particularly glucose, implying that there are additional transduction mechanisms and/or taste receptors responsive to sugars and leading to the preference for sugars. In addition, analysis of the nerve recording and behavioral data obtained from T1r3 KO mice shows that T1r3 plays an important role in umami taste responses but is not the only umami taste receptor. In vitro, cells heterologously expressing T1r1 and T1r3 respond to glutamate and other umami compounds, particularly in the presence of IMP (13, 14). In vivo, the CT and GL nerves of B6 wild-type mice both respond to glutamate, although there are key differences between these two nerves in their responses to MSG and other umami compounds, suggesting that multiple receptors might be involved in MSG detection (17, 20). T1r3 is essential for CT responses to MSG+IMP and is probably also involved in CT responses to MSG. However, T1r3 does not play a role in GL responses to either MSG alone or MSG+IMP, indicating that other receptors [such as taste-mGluR4 (22)] mediate GL responses to these particular umami compounds. Perhaps taste-mGluR4 underlies the avoidance response elicited by high concentrations of MSG.

    Supporting Online Material

    www.sciencemag.org/cgi/content/full/1087155/DC1

    Materials and Methods

    Figs. S1 and S2

    References

    References and Notes

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