We found the expression of CRLR in both T1R3-positive cells and in CA4-positive cells. On the other hand, most RAMP2-expressing taste cells were T1R3-positive but not CA4-positive (Figure 1, Table 2). This is consistent with previous RNA sequencing data showing the expression of CRLR in both type II and in type III cells and the expression of RAMP2 in the type II cells of mouse taste buds [23,24]. These results indicate that at the very least, an ADM receptor consisting of CRLR and RAMP2 is preferentially expressed in T1R3-positive taste cells. CRLR and RAMP2 or RAMP3 form the ADM receptor, whereas CRLR and RAMP1 form the CGRP receptor [35]. The expression of CGRP was reported in the sensory neuron innervating the CV in rats [36]. Furthermore, CGRP evoked serotonin (5-HT) secretion from presynaptic cells (type III taste cells) and released 5-HT-provided negative paracrine feedback onto receptor cells (type II taste cells) via the activation of 5-HT_1A receptors, subsequently inducing a reduction in ATP secretion [37]. Therefore, the CRLR expressed on CA4-positive taste cells may function as a component of the CGRP receptor. This should be investigated in future studies.

The intravenous injection of ADM enhanced CT nerve responses to sugars but not to artificial sweeteners in B6 mice (Figure 2A–D). The half-life of circulating ADM is reported to be 16~22 min [38]. Consistent with this report, the enhancing effect of ADM on CT nerve responses to sugars was observed from 5 min after an i.v. injection of ADM, reached its maximum level approximately 10 min after, and was maintained until 30 min after, when the responses started to recover (Figure 2B). A similar time course of the effect of ADM after an i.v. injection was reported in in vivo experiments investigating its effects on vasodilation [39] and cardiovascular changes [40] in rats. The enhancing effects of ADM on CT nerve responses to sugars were reduced via pretreatment with the ADM receptor antagonist AM (22–52) (Figure 3A–D), indicating that these enhancing effects of ADM were mediated by ADM receptors. In our experiments, CRLR and RAMP2 proteins were likely to be expressed in T1R3-positive taste cells, which may play a key role in detecting sugars in the oral cavity. Therefore, ADM may affect T1R3-positive cells expressing both CRLR and RAMP2 to enhance the sugar responses of these cells.

Enhancing effects of ADM on responses to sugars were also found in T1R3-KO mice (Figure 4A,B). Moreover, the SGLT1 inhibitor phlorizin reduced the enhancing effects of ADM on sugars and sugar-NaCl mixtures in T1R3-KO mice (Figure 5A,B). We had reported that SGLT1 may mediate the T1R-independent sweet taste of sugars [8]. Taking these data together, SGLT1 may be a key component for the enhancement of sugar responses by ADM. To support this hypothesis, the apical uptake of a fluorescent D-glucose derivative in taste cells was also increased by ADM treatment, and these effects were almost completely blocked by AM (22–52) or phlorizin pretreatment (Figure 6). ADM injection also increased the mRNA expression level of SGLT1 but not of other glucose transporters or T1R3 in FP and CV taste buds, and AM (22–52) inhibited this effect (Figure 7).

How does ADM modulate SGLT1 expression and increase the uptake of glucose into taste cells? In many types of cells, both ADM and CGRP receptors are coupled with the stimulatory G protein G_αs, which activates adenylate cyclase and increases intracellular cyclic adenosine monophosphate (cAMP) levels [41]. Taste cells are also reported to express several isoforms of adenylyl cyclase [42] and phosphodiesterase [43], which synthesize and degrade cAMP, respectively. Cyclic AMP has been shown to increase the amiloride-sensitive Na⁺ current in isolated hamster taste bud cells [42] and the CT nerve response to NaCl in rats [44]. These enhancements may occur via cAMP pathway activation, leading to the recruitment of an intracellular pool of the epithelial sodium channel (ENaC) to the plasma membrane [44]. Angiotensin II (Ang II) is a well-known vasopressor hormone, and its receptor is also a G-protein-coupled receptor (GPCR). Ang II stimulates early proximal tubule bicarbonate absorption by decreasing the intracellular cAMP level via Ang II receptor activation in rats [45]. On the other hand, in the kidney, aldosterone (Aldo), also a vasopressor hormone, binds to the cytosolic mineralocorticoid receptor, and the Aldo/receptor complex is then translocated to the nucleus, leading to ENaC mRNA transcription and then ENaC protein trafficking to the cell surface [46]. Furthermore, cAMP and serum and glucocorticoid-inducible kinase, a downstream molecule of Aldo, is reported to increase ENaC protein trafficking [47]. We previously reported that Ang II reduced taste sensitivities for NaCl in mice [48]. Conversely, it was demonstrated that Aldo enhanced taste sensitivities for NaCl in rats [49]. These reductions and enhancements of responses to NaCl may have occurred via the modulation of cAMP through the activation of each hormone’s receptors [48,49], resulting in the downregulation or upregulation of ENaC on the plasma membrane. Focusing on SGLT1, the activation of the cAMP-PKA pathway upregulated SGLT1 expression in the intestines of sheep [50,51]. In rat intestines, ADM increased SGLT1 protein expression and α-methylglucoside absorption [25]. Therefore, the activation of the cAMP pathway via ADM receptor may upregulate SGLT1 expression. Taken together, such mechanisms may be involved in the enhancement of CT nerve responses to sugars (Figure 2D), 2-NBDG uptake (Figure 6), and SGLT1 mRNA expression levels (Figure 7) within 30 min of ADM administration. Although there were many reports demonstrating the contributions of the cAMP-PKA pathway to the regulation of SGLT1 expression, its time course varied greatly from 20 min [52] to several hours [53]. These differences may result from differences in the cell lines used, in vivo vs. in vitro approaches, or experimental designs. To clarify the possibility of SGLT1 recruitment via ADM receptor activation in taste cells, further studies are needed.

At the peripheral taste tissue, multiple sugar sensors and information pathways may play differential roles that work in combination to support food intake and energy homeostasis. For example, metabolic sensor K_ATP channels are coupled with the glucose transporter pathway in taste cells and play a crucial role in CPIR after oral stimulation with sugars [10,11]. The anorexigenic mediator leptin suppressed sweet taste sensitivities, and its suppression of sweet responses was mediated by the leptin receptor Ob-Rb [12,13,14,15] and by phosphoinositide 3-kinase in the K_ATP channel pathway, which is expressed on T1R3-positive taste cells [54]. Conversely, 2-arachidonylglycerol (2-AG), one of the endocannabinoids known as orexigenic mediators, selectively enhanced sweet taste responses via the expression of CB1 on T1R3-positive taste cells [16]. Interestingly, 2-AG synthase diacylglycerol lipase α and β were also expressed in T1R3-positive taste cells in mice [17]. The major pathway of 2-AG synthesis is reported to be the GPCR–phospholipase C–diacylglycerol pathway [55,56]. These data imply that a T1R-dependent pathway may receive autocrine feedback via 2-AG [17]. Circulating leptin but not endocannabinoids may act as a dominant modulator for sweet taste in lean wild-type mice [17]. Therefore, endocannabinoids may function as more effective sweet taste modulators under the conditions of deficient leptin signaling by increased 2-AG synthesis in taste tissue [17]. Leptin and endocannabinoids may contribute to energy intake by modulating sweet taste sensitivities competitively via T1R-dependent and T1R-independent sweet taste signaling pathways [17]. Focusing on the neural taste pathway, single-fiber recordings of CT nerve responses in wild-type mice revealed that S-type fibers showing maximal responses to sucrose could be classified into T1R-dependent (phlorizin-insensitive), Glc, and mixed (SGLTs and T1Rs) types [8]. Interestingly, a subset of S-type fibers also respond to glutamate with inosine-5′-monophosphate [57] and to the long-chain fatty acids linoleic acid and oleic acid [58]. Therefore, there is a group of fibers that respond to three major nutrients, suggesting that the taste cells connecting to this subset of S-type fibers may function to find energy sources [58]. Taking into consideration the modulation systems of sweet taste sensitivity between T1R-dependent and/or T1R-independent pathways, as mentioned above, ADM may also contribute to caloric sensing and food intake via the T1R-independent sweet taste pathway. Interestingly, an intracerebroventricular injection of ADM has been proposed to regulate food intake in previous studies with animal models [59,60,61]. However, this should be verified by future studies because detailed mechanisms within and between peripheral and central taste sensing systems remain unresolved.

In conclusion, we have demonstrated that an SGLT1-dependent sugar taste pathway may be the target of ADM for the enhancement of sugar information. ADM receptor components were expressed on T1R3-positive taste cells in mouse FP. An ADM injection enhanced CT nerve responses to sugars but not to artificial sweeteners or other tastants in both wild-type and in T1R3-KO mice, facilitated glucose uptake in taste cells, and increased SGLT1 mRNA expression. These effects of ADM injection were reduced via a pretreatment with the ADM receptor inhibitor AM (22–52), indicating that these effects of ADM were mediated via the ADM receptor. Moreover, ADM enhanced synergistic responses to binary mixtures of sugars with 10 mM of NaCl, and this effect was blocked by pretreatment with the SGLT1 inhibitor phlorizin, indicating that SGLT1 is a key component for the enhancement of sugar responses by ADM. Taken together, our results indicate that ADM may contribute to caloric sensing and food intake by enhancing gustatory nerve responses to sugars via a T1R-independent pathway that utilizes an SGLT glucose transporter.