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Leptin is an anorexigenic mediator that reduces food intake by acting on hypothalamic receptor Ob-Rb. In contrast, endocannabinoids are orexigenic mediators that act via cannabinoid CB1 receptors in hypothalamus, limbic forebrain, and brainstem. In the peripheral taste system, leptin administration selectively inhibits behavioural, taste nerve and taste cell responses to sweet compounds. Opposing the action of leptin, endocannabinoids enhance sweet taste responses. However, potential roles of endogenous leptin and endocannabinoids in sweet taste remain unclear. Here, we used pharmacological antagonists (Ob-Rb: L39A/D40A/F41A (LA), CB1: AM251) and examined the effects of their blocking activation of endogenous leptin and endocannabinoid signalling on taste responses in lean control, leptin receptor deficient db/db, and diet-induced obese (DIO) mice. Lean mice exhibited significant increases in chorda tympani (CT) nerve responses to sweet compounds after LA administration, while they showed no significant changes in CT responses after AM251. In contrast, db/db mice showed clear suppression of CT responses to sweet compounds after AM251, increased endocannabinoid (2-arachidonoyl-sn-glycerol (2-AG)) levels in the taste organ, and enhanced expression of a biosynthesizing enzyme (diacylglycerol lipase α (DAGLα)) of 2-AG in taste cells. In DIO mice, the LA effect was gradually decreased and the AM251 effect was increased during the course of obesity. Taken together, our results suggest that circulating leptin, but not local endocannabinoids, may be a dominant modulator for sweet taste in lean mice; however, endocannabinoids may become more effective modulators of sweet taste under conditions of deficient leptin signalling, possibly due to increased production of endocannabinoids in taste tissue. Key points Potential roles of endogenous leptin and endocannabinoids in sweet taste were examined by using pharmacological antagonists and mouse models including leptin receptor deficient (db/db) and diet-induced obese (DIO) mice. Chorda tympani (CT) nerve responses of lean mice to sweet compounds were increased after administration of leptin antagonist (LA) but not affected by administration of cannabinoid receptor antagonist (AM251). db/db mice showed clear suppression of CT responses to sweet compounds after AM251, increased endocannabinoid levels in the taste organ, and enhanced expression of a biosynthesizing enzyme of endocannabinoids in taste cells. The effect of LA was gradually decreased and that of AM251 was increased during the course of obesity in DIO mice. These findings suggest that circulating leptin, but not local endocannabinoids, is a dominant modulator for sweet taste in lean mice and endocannabinoids become more effective modulators of sweet taste under conditions of deficient leptin signalling. Introduction Taste is primarily devoted to the selection of foods and required nutrients, and the avoidance of potentially harmful compounds. Sweet sensing is thought to be essential for the detection of carbohydrate sources of calories. Sensory input from sweet sensing cells to the brain may serve as a signal that can provoke palatability of food to stimulate food intake, and facilitate secretion of insulin in the pancreas and other factors in the gut involved in regulating nutrient absorption and energy homeostasis. Recent studies reveal that the peripheral sweet-sensing system is modulated by the anorexigenic mediator leptin (Kawai et al. 2000) and the orexigenic endocannabinoids 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (N-arachidonoylethanolamine (AEA) (Yoshida et al. 2010). Leptin, an adipocyte-derived hormone, primarily acts on functional leptin receptors (Ob-Rb) in the hypothalamus and reduces food intake, increases energy expenditure, and regulates body weight (Friedman & Halaas, 1998; Friedman 2004). The db/db mice have a point mutation of the db gene, lack functional leptin receptors, and are hyperphagic, massively obese and diabetic (Lee et al. 1996). In the peripheral taste system, taste cells express Ob-Rb and administration of recombinant leptin selectively suppresses taste cell, taste nerve and behavioural responses to sweet compounds without affecting responses to other basic taste stimuli (salty, sour, bitter and umami compounds) in lean control mice (Kawai et al. 2000; Shigemura et al. 2004; Yoshida et al. 2013). The db/db mice show no such leptin inhibition on sweet taste responses, but instead exhibit greater gustatory neural sensitivities (Ninomiya et al. 1995, 1998; Sako et al. 1996) and higher behavioural preferences (Ninomiya et al. 1995) for various sweet compounds compared with lean control mice. Endocannabinoids act on cannabinoid receptor 1 (CB1 receptor) in the hypothalamus and limbic forebrain to induce appetite (Jamshidi & Taylor, 2001; Cota et al. 2003) and stimulate food intake, which opposes the action of leptin. Systemic administration of exogenous cannabinoids and endocannabinoids in rodents causes hyperphagia (Williams & Kirkham, 1999) and increases the preference for palatable substances, such as sucrose solution and sweetened food pellets (Higgs et al. 2003; Jarrett et al. 2005). Infusions of 2-AG into the pontine parabrachial nucleus, which contains third order gustatory neurons, increase intake of palatable foods, including sucrose pellets, without affecting the intake of normal chow (DiPatrizio & Simansky, 2008), suggesting that endocannabinoids may be related to hedonic aspects of sweet taste. In the peripheral taste system, comparable with their central action, systemic administration of 2-AG or AEA selectively increased taste cell, nerve and behavioural responses to sweeteners without affecting responses to salty, sour, bitter and umami compounds (Yoshida et al. 2010). Mice genetically lacking CB1 receptors showed no such enhancement of sweet taste responses by endocannabinoids and the sweet-enhancing effect was prevented by a CB1 antagonist (Yoshida et al. 2010), indicating that the effect may be mediated by CB1 receptors. Reciprocal regulation of peripheral sweet taste reception by endocannabinoids and leptin may, thus, contribute to their opposing actions on food intake and energy homeostasis. In our previous studies (Kawai et al. 2000; Yoshida et al. 2010), however, reciprocal modulation of sweet taste responses by leptin and endocannabinoids was found only in separate experiments after systemic administration of exogenous leptin and endocannabinoids to mice. Therefore, it remains unclear to what extent endogenous leptin or endocannabinoids tonically affect peripheral taste responsiveness in mice. In the hypothalamus, leptin inhibits production of endocannabinoids (Jo et al. 2005). Conversely, hypothalamic endocannabinoids are increased in genetically obese rodents lacking functional leptin (ob/ob mice) and leptin receptors (db/db mice and Zucker rats; Di Marzo et al. 2001). These studies suggest that endocannabinoids are normally under negative control by leptin, and might be involved in the hyperphagia and obesity that accompany defects in leptin signalling. A potential site for the interaction between leptin and endocannabinoids is reported in lateral hypothalamic neurons, where leptin inhibits voltage-gated calcium channels, preventing the influx of calcium into cells and blocking endocannabinoid release and synthesis (Jo et al. 2005), a mechanism that contributes to the appetite-suppressing effect of leptin. This evidence raises the possibility that such interaction between leptin and endocannabinds could also be involved in modulation of sweet responsiveness of taste cells and the sweet taste modulator could be switched from leptin in lean mice to endocannabinoid in obese mice with defects in leptin signalling. To answer these questions, we first investigated the effects of pharmacological inhibition of leptin or endocannabinoid receptors on peripheral taste responses in lean control mice with and without food deprivation, and in db/db mice. We treated mice with a leptin receptor antagonist, the leptin triple mutant L39A/D40A/F41A (LA), which is reported to increase food intake of normal chow-fed rats when infused into the lateral ventricle (Zhang et al. 2007), and the CB1 antagonist AM251, which is shown following systemic administration in mice to decrease the intake of a high-fat diet (Hildebrandt et al. 2003). Furthermore, we investigated the molecular basis for endocannabinoid actions on the taste organ. Finally, we investigated a proposed switch of the sweet taste modulator from leptin to endocannabinoids by comparing LA and AM251 effects on sweet taste responses at different stages of development of diet-induced obese (DIO) mice.

Key points The taste receptor T1R1 + T1R3 heterodimer and metabotropic glutamate receptors (mGluR) may function as umami taste receptors. Here, we used mGluR4 knockout (mGluR4-KO) mice and examined the function of mGluR4 in peripheral taste responses of mice. The mGluR4-KO mice showed reduced responses to glutamate and l-AP4 (mGluR4 agonist) in the chorda tympani and glossopharyngeal nerves without affecting responses to other taste stimuli. Residual glutamate responses in mGluR4-KO mice were suppressed by gurmarin (T1R3 blocker) and AIDA (group I mGluR antagonist). The present study not only provided functional evidence for the involvement of mGluR4 in umami taste responses, but also suggested contributions of T1R1 + T1R3 and mGluR1 receptors in glutamate responses. Abstract Umami taste is elicited by l-glutamate and some other amino acids and is thought to be initiated by G-protein-coupled receptors. Proposed umami receptors include heterodimers of taste receptor type 1, members 1 and 3 (T1R1 + T1R3), and metabotropic glutamate receptors 1 and 4 (mGluR1 and mGluR4). Accumulated evidences support the involvement of T1R1 + T1R3 in umami responses in mice. However, little is known about the in vivo function of mGluR in umami taste. Here, we examined taste responses of the chorda tympani (CT) and the glossopharyngeal (GL) nerves in wild-type mice and mice genetically lacking mGluR4 (mGluR4-KO). Our results indicated that compared to wild-type mice, mGluR4-KO mice showed significantly smaller gustatory nerve responses to glutamate and l-(+)-2-amino-4-phosphonobutyrate (an agonist for group III mGluR) in both the CT and GL nerves without affecting responses to other taste stimuli. Residual glutamate responses in mGluR4-KO mice were not affected by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (an antagonist for group III mGluR), but were suppressed by gurmarin (a T1R3 blocker) in the CT and (RS)-1-aminoindan-1,5-dicarboxylic acid (an antagonist for group I mGluR) in the CT and GL nerve. In wild-type mice, both quisqualic acid (an agonist for group I mGluR) and l-(+)-2-amino-4-phosphonobutyrate elicited gustatory nerve responses and these responses were suppressed by addition of (RS)-1-aminoindan-1,5-dicarboxylic acid and (RS)-alpha-cyclopropyl-4-phosphonophenylglycine, respectively. Collectively, the present study provided functional evidences for the involvement of mGluR4 in umami taste responses in mice. The results also suggest that T1R1 + T1R3 and mGluR1 are involved in umami taste responses in mice. Thus, umami taste would be mediated by multiple receptors.

Key points • The taste receptor heterodimer T1R1 + T1R3, metabotropic glutamate receptors (mGluRs) and/or their variants may function as umami taste receptors. • Here, we used newly developed T1R1−/− mice and examined the role of T1R1 and mGluRs in taste detection. • The T1R1−/− mice exhibited seriously diminished synergistic responses to glutamate and inosine monophosphate but not to glutamate alone and significantly smaller responses to sweeteners. • Addition of mGluR antagonists significantly inhibited responses to glutamate in both T1R1−/− and heterozygous T1R1+/− mice. • Taken together, these results suggest that T1R1 mainly contributes to umami taste synergism and partly to sweet sensitivity, while mGluRs are involved in the detection of umami compounds. Abstract The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1−/−) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1−/− mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1−/− mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1+/− mice responded to sweet and umami compounds, whereas those in T1R1−/− mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1−/− than in T1R1+/− mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1−/− and T1R1+/− mice. Conditioned taste aversion tests demonstrated that both T1R1−/− and T1R1+/− mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds.

The distinctive umami taste elicited by l-glutamate and some other amino acids is thought to be initiated by G-protein-coupled receptors. Proposed umami receptors include heteromers of taste receptor type 1, members 1 and 3 (T1R1+T1R3), and metabotropic glutamate receptors 1 and 4 (mGluR1 and mGluR4). Multiple lines of evidence support the involvement of T1R1+T1R3 in umami responses of mice. Although several studies suggest the involvement of receptors other than T1R1+T1R3 in umami, the identity of those receptors remains unclear. Here, we examined taste responsiveness of umami-sensitive chorda tympani nerve fibres from wild-type mice and mice genetically lacking T1R3 or its downstream transduction molecule, the ion channel TRPM5. Our results indicate that single umami-sensitive fibres in wild-type mice fall into two major groups: sucrose-best (S-type) and monopotassium glutamate (MPG)-best (M-type). Each fibre type has two subtypes; one shows synergism between MPG and inosine monophosphate (S1, M1) and the other shows no synergism (S2, M2). In both T1R3 and TRPM5 null mice, S1-type fibres were absent, whereas S2-, M1- and M2-types remained. Lingual application of mGluR antagonists selectively suppressed MPG responses of M1- and M2-type fibres. These data suggest the existence of multiple receptors and transduction pathways for umami responses in mice. Information initiated from T1R3-containing receptors may be mediated by a transduction pathway including TRPM5 and conveyed by sweet-best fibres, whereas umami information from mGluRs may be mediated by TRPM5-independent pathway(s) and conveyed by glutamate-best fibres.

Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.

Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.