GPRC6A as a novel kokumi receptor responsible for enhanced taste preferences by ornithine

Reviewer #1 (Public review):

Summary:

This paper contains what could be described as a "classic" approach towards evaluating a novel taste stimuli in an animal model, including standard behavioral tests (some with nerve transections), taste nerve physiology, and immunocytochemistry of the tongue. The stimulus being tested is ornithine, from a class of stimuli called "kokumi", which are stimuli that enhance other canonical tastes, increasing essentially the hedonic attributes of these other stimuli; the mechanism for ornithine detection is thought to be GPRC6A receptors expressed in taste cells. The authors showed evidence for this in an earlier paper with mice; this paper evaluates ornithine taste in a rat model.

Strengths:

The data show the effects of ornithine on taste: in two-bottle and briefer intake tests, adding ornithine results in a higher intake of most, but not all, stimuli tests. Bilateral nerve cuts or the addition of GPRC6A antagonists decrease this effect. Small effects of ornithine are shown in whole-nerve recordings.

Weaknesses:

The conclusion seems to be that the authors have found evidence for ornithine acting as a taste modifier through the GPRC6A receptor expressed on the anterior tongue. It is hard to separate their conclusions from the possibility that any effects are additive rather than modulatory. Animals did prefer ornithine to water when presented by itself. Additionally, the authors refer to evidence that ornithine is activating the T1R1-T1R3 amino acid taste receptor, possibly at higher concentrations than they use for most of the study, although this seems speculative. It is striking that the largest effects on taste are found with the other amino acid (umami) stimuli, leading to the possibility that these are largely synergistic effects taking place at the tas1r receptor heterodimer.

Reviewer #2 (Public review):

Summary:

The authors used rats to determine the receptor for a food-related perception (kokumi) that has been characterized in humans. They employ a combination of behavioral, electrophysiological, and immunohistochemical results to support their conclusion that ornithine-mediated kokumi effects are mediated by the GPRC6A receptor. They complemented the rat data with some human psychophysical data. I find the results intriguing, but believe that the authors overinterpret their data.

Strengths:

The authors examined a new and exciting taste enhancer (ornithine). They used a variety of experimental approaches in rats to document the impact of ornithine on taste preference and peripheral taste nerve recordings. Further, they provided evidence pointing to a potential receptor for ornithine.

Weaknesses:

The authors have not established that the rat is an appropriate model system for studying kokumi. Their measurements do not provide insight into any of the established effects of kokumi on human flavor perception. The small study on humans is difficult to compare to the rat study because the authors made completely different types of measurements. Thus, I think that the authors need to substantially scale back the scope of their interpretations. These weaknesses diminish the likely impact of the work on the field of flavor perception.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to investigate whether GPRC6A mediates kokumi taste initiated by the amino acid L-ornithine. They used Wistar rats, a standard laboratory strain, as the primary model and also performed an informative taste test in humans, in which miso soup was supplemented with various concentrations of L-ornithine. The findings are valuable and overall the evidence is solid. L-Ornithine should be considered to be a useful test substance in future studies of kokumi taste and the class C G protein-coupled receptor known as GPRC6A (C6A) along with its homolog, the calcium-sensing receptor (CaSR) should be considered candidate mediators of kokumi taste.

Strengths:

The overall experimental design is solid based on two bottle preference tests in rats. After determining the optimal concentration for L-Ornithine (1 mM) in the presence of MSG, it was added to various tastants, including inosine 5'-monophosphate; monosodium glutamate (MSG); mono-potassium glutamate (MPG); intralipos (a soybean oil emulsion); sucrose; sodium chloride (NaCl); citric acid and quinine hydrochloride. Robust effects of ornithine were observed in the cases of IMP, MSG, MPG, and sucrose, and little or no effects were observed in the cases of sodium chloride, citric acid, and quinine HCl. The researchers then focused on the preference for Ornithine-containing MSG solutions. The inclusion of the C6A inhibitors Calindol (0.3 mM but not 0.06 mM) or the gallate derivative EGCG (0.1 mM but not 0.03 mM) eliminated the preference for solutions that contained Ornithine in addition to MSG. The researchers next performed transections of the chord tympani nerves (with sham operation controls) in anesthetized rats to identify the role of the chorda tympani branches of the facial nerves (cranial nerve VII) in the preference for Ornithine-containing MSG solutions. This finding implicates the anterior half-two thirds of the tongue in ornithine-induced kokumi taste. They then used electrical recordings from intact chorda tympani nerves in anesthetized rats to demonstrate that ornithine enhanced MSG-induced responses following the application of tastants to the anterior surface of the tongue. They went on to show that this enhanced response was insensitive to amiloride, selected to inhibit 'salt tastant' responses mediated by the epithelial Na+ channel, but eliminated by Calindol. Finally, they performed immunohistochemistry on sections of rat tongue demonstrating C6A positive spindle-shaped cells in fungiform papillae that partially overlapped in its distribution with the IP3 type-3 receptor, used as a marker of Type-II cells, but not with (i) gustducin, the G protein partner of Tas1 receptors (T1Rs), used as a marker of a subset of type-II cells; or (ii) 5-HT (serotonin) and Synaptosome-associated protein 25 kDa (SNAP-25) used as markers of Type-III cells.

Weaknesses:

The researchers undertook what turned out to be largely confirmatory studies in rats with respect to their previously published work on Ornithine and C6A in mice (Mizuta et al Nutrients 2021).

The authors point out that animal models pose some difficulties of interpretation in studies of taste and raise the possibility in the Discussion that umami substances may enhance the taste response to ornithine (Line 271, Page 9). They miss an opportunity to outline the experimental results from the study that favor their preferred interpretation that ornithine is a taste enhancer rather than a tastant.

At least two other receptors in addition to C6A might mediate taste responses to ornithine: (i) the CaSR, which binds and responds to multiple L-amino acids (Conigrave et al, PNAS 2000), and which has been previously reported to mediate kokumi taste (Ohsu et al., JBC 2010) as well as responses to Ornithine (Shin et al., Cell Signaling 2020); and (ii) T1R1/T1R3 heterodimers which also respond to L-amino acids and exhibit enhanced responses to IMP (Nelson et al., Nature 2001). While the experimental results as a whole favor the authors' interpretation that C6A mediates the Ornithine responses, they do not make clear either the nature of the 'receptor identification problem' in the Introduction or the way in which they approached that problem in the Results and Discussion sections. It would be helpful to show that a specific inhibitor of the CaSR failed to block the ornithine response. In addition, while they showed that C6A-positive cells were clearly distinct from gustducin-positive, and thus T1R-positive cells, they missed an opportunity to clearly differentiate C6A-expressing taste cells and CaSR-expressing taste cells in the rat tongue sections.

It would have been helpful to include a positive control kokumi substance in the two-bottle preference experiment (e.g., one of the known gamma-glutamyl peptides such as gamma-glu-Val-Gly or glutathione), to compare the relative potencies of the control kokumi compound and Ornithine, and to compare the sensitivities of the two responses to C6A and CaSR inhibitors.

The results demonstrate that enhancement of the chorda tympani nerve response to MSG occurs at substantially greater Ornithine concentrations (10 and 30 mM) than were required to observe differences in the two bottle preference experiments (1.0 mM; Figure 2). The discrepancy requires careful discussion and if necessary further experiments using the two-bottle preference format.

Author response:

Reviewer #1 (Public review):

Summary:

This paper contains what could be described as a "classic" approach towards evaluating a novel taste stimuli in an animal model, including standard behavioral tests (some with nerve transections), taste nerve physiology, and immunocytochemistry of the tongue. The stimulus being tested is ornithine, from a class of stimuli called "kokumi", which are stimuli that enhance other canonical tastes, increasing essentially the hedonic attributes of these other stimuli; the mechanism for ornithine detection is thought to be GPRC6A receptors expressed in taste cells. The authors showed evidence for this in an earlier paper with mice; this paper evaluates ornithine taste in a rat model.

Strengths:

The data show the effects of ornithine on taste: in two-bottle and briefer intake tests, adding ornithine results in a higher intake of most, but not all, stimuli tests. Bilateral nerve cuts or the addition of GPRC6A antagonists decrease this effect. Small effects of ornithine are shown in whole-nerve recordings.

Weaknesses:

The conclusion seems to be that the authors have found evidence for ornithine acting as a taste modifier through the GPRC6A receptor expressed on the anterior tongue. It is hard to separate their conclusions from the possibility that any effects are additive rather than modulatory. Animals did prefer ornithine to water when presented by itself. Additionally, the authors refer to evidence that ornithine is activating the T1R1-T1R3 amino acid taste receptor, possibly at higher concentrations than they use for most of the study, although this seems speculative. It is striking that the largest effects on taste are found with the other amino acid (umami) stimuli, leading to the possibility that these are largely synergistic effects taking place at the tas1r receptor heterodimer.

We would like to thank Reviewer #1 for the valuable comments. Our basis for considering ornithine as a taste modifier stems from our observation that a low concentration of ornithine (1 mM), which does not elicit a preference on its own, enhances the preference for umami substances, sucrose, and soybean oil through the activation of the GPRC6A receptor. Notably, this receptor is not typically considered a taste receptor. The reviewer suggested that the enhancement of umami taste might be due to potentiation occurring at the TAS1R receptor heterodimer. However, we propose that a different mechanism may be at play, as an antagonist of GPRC6A almost completely abolished this enhancement. In the revised manuscript, we will endeavor to provide additional information on the role of ornithine as a taste modifier acting through the GPRC6A receptor.

Reviewer #2 (Public review):

Summary:

The authors used rats to determine the receptor for a food-related perception (kokumi) that has been characterized in humans. They employ a combination of behavioral, electrophysiological, and immunohistochemical results to support their conclusion that ornithine-mediated kokumi effects are mediated by the GPRC6A receptor. They complemented the rat data with some human psychophysical data. I find the results intriguing, but believe that the authors overinterpret their data.

Strengths:

The authors examined a new and exciting taste enhancer (ornithine). They used a variety of experimental approaches in rats to document the impact of ornithine on taste preference and peripheral taste nerve recordings. Further, they provided evidence pointing to a potential receptor for ornithine.

Weaknesses:

The authors have not established that the rat is an appropriate model system for studying kokumi. Their measurements do not provide insight into any of the established effects of kokumi on human flavor perception. The small study on humans is difficult to compare to the rat study because the authors made completely different types of measurements. Thus, I think that the authors need to substantially scale back the scope of their interpretations. These weaknesses diminish the likely impact of the work on the field of flavor perception.

We would like to thank Reviewer #2 for the valuable comments and suggestions. Regarding the question of whether the rat is an appropriate model system for studying kokumi, we have chosen this species for several reasons: it is readily available as a conventional experimental model for gustatory research; the calcium-sensing receptor (CaSR), known as the kokumi receptor, is expressed in taste bud cells; and prior research has demonstrated the use of rats in kokumi studies involving gamma Glu-Val-Gly (Yamamoto and Mizuta, Chem. Senses, 2022). We acknowledge that fundamentally different types of measurements were conducted in the human psychophysical study and the rat study. Kokumi can indeed be assessed and expressed in humans; however, we do not currently have the means to confirm that animals experience kokumi in the same way that humans do. Therefore, human studies are necessary to evaluate kokumi, a conceptual term denoting enhanced flavor, while animal studies are needed to explore the potential underlying mechanisms of kokumi. We believe that a combination of both human and animal studies is essential, as is the case with research on sugars. While sugars are known to elicit sweetness, it is unclear whether animals perceive sweetness identically to humans, even though they exhibit a strong preference for sugars. In the revised manuscript, we will incorporate additional information to address the comments raised by the reviewer. We will also carefully review and revise our previous statements to ensure accuracy and clarity.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to investigate whether GPRC6A mediates kokumi taste initiated by the amino acid L-ornithine. They used Wistar rats, a standard laboratory strain, as the primary model and also performed an informative taste test in humans, in which miso soup was supplemented with various concentrations of L-ornithine. The findings are valuable and overall the evidence is solid. L-Ornithine should be considered to be a useful test substance in future studies of kokumi taste and the class C G protein-coupled receptor known as GPRC6A (C6A) along with its homolog, the calcium-sensing receptor (CaSR) should be considered candidate mediators of kokumi taste.

Strengths:

The overall experimental design is solid based on two bottle preference tests in rats. After determining the optimal concentration for L-Ornithine (1 mM) in the presence of MSG, it was added to various tastants, including inosine 5'-monophosphate; monosodium glutamate (MSG); mono-potassium glutamate (MPG); intralipos (a soybean oil emulsion); sucrose; sodium chloride (NaCl); citric acid and quinine hydrochloride. Robust effects of ornithine were observed in the cases of IMP, MSG, MPG, and sucrose, and little or no effects were observed in the cases of sodium chloride, citric acid, and quinine HCl. The researchers then focused on the preference for Ornithine-containing MSG solutions. The inclusion of the C6A inhibitors Calindol (0.3 mM but not 0.06 mM) or the gallate derivative EGCG (0.1 mM but not 0.03 mM) eliminated the preference for solutions that contained Ornithine in addition to MSG. The researchers next performed transections of the chord tympani nerves (with sham operation controls) in anesthetized rats to identify the role of the chorda tympani branches of the facial nerves (cranial nerve VII) in the preference for Ornithine-containing MSG solutions. This finding implicates the anterior half-two thirds of the tongue in ornithine-induced kokumi taste. They then used electrical recordings from intact chorda tympani nerves in anesthetized rats to demonstrate that ornithine enhanced MSG-induced responses following the application of tastants to the anterior surface of the tongue. They went on to show that this enhanced response was insensitive to amiloride, selected to inhibit 'salt tastant' responses mediated by the epithelial Na+ channel, but eliminated by Calindol. Finally, they performed immunohistochemistry on sections of rat tongue demonstrating C6A positive spindle-shaped cells in fungiform papillae that partially overlapped in its distribution with the IP3 type-3 receptor, used as a marker of Type-II cells, but not with (i) gustducin, the G protein partner of Tas1 receptors (T1Rs), used as a marker of a subset of type-II cells; or (ii) 5-HT (serotonin) and Synaptosome-associated protein 25 kDa (SNAP-25) used as markers of Type-III cells.

Weaknesses:

The researchers undertook what turned out to be largely confirmatory studies in rats with respect to their previously published work on Ornithine and C6A in mice (Mizuta et al Nutrients 2021).

The authors point out that animal models pose some difficulties of interpretation in studies of taste and raise the possibility in the Discussion that umami substances may enhance the taste response to ornithine (Line 271, Page 9). They miss an opportunity to outline the experimental results from the study that favor their preferred interpretation that ornithine is a taste enhancer rather than a tastant.

At least two other receptors in addition to C6A might mediate taste responses to ornithine: (i) the CaSR, which binds and responds to multiple L-amino acids (Conigrave et al, PNAS 2000), and which has been previously reported to mediate kokumi taste (Ohsu et al., JBC 2010) as well as responses to Ornithine (Shin et al., Cell Signaling 2020); and (ii) T1R1/T1R3 heterodimers which also respond to L-amino acids and exhibit enhanced responses to IMP (Nelson et al., Nature 2001). While the experimental results as a whole favor the authors' interpretation that C6A mediates the Ornithine responses, they do not make clear either the nature of the 'receptor identification problem' in the Introduction or the way in which they approached that problem in the Results and Discussion sections. It would be helpful to show that a specific inhibitor of the CaSR failed to block the ornithine response. In addition, while they showed that C6A-positive cells were clearly distinct from gustducin-positive, and thus T1R-positive cells, they missed an opportunity to clearly differentiate C6A-expressing taste cells and CaSR-expressing taste cells in the rat tongue sections.

It would have been helpful to include a positive control kokumi substance in the two-bottle preference experiment (e.g., one of the known gamma-glutamyl peptides such as gamma-glu-Val-Gly or glutathione), to compare the relative potencies of the control kokumi compound and Ornithine, and to compare the sensitivities of the two responses to C6A and CaSR inhibitors.

The results demonstrate that enhancement of the chorda tympani nerve response to MSG occurs at substantially greater Ornithine concentrations (10 and 30 mM) than were required to observe differences in the two bottle preference experiments (1.0 mM; Figure 2). The discrepancy requires careful discussion and if necessary further experiments using the two-bottle preference format.

We would like to thank Reviewer #3 for the valuable comments and helpful suggestions. We propose that ornithine has two stimulatory actions: one acting on GPRC6A, particularly at lower concentrations, and another on amino acid receptors such as T1R1/T1R3 at higher concentrations. Consequently, ornithine is not preferable at lower concentrations but becomes preferable at higher concentrations. For our study on kokumi, we used a low concentration (1 mM) of ornithine. The possibility mentioned in the Discussion that 'the umami substances may enhance the taste response to ornithine' is entirely speculative. We will reconsider including this description in the revised version. As the reviewer suggested, in addition to GPRC6A, ornithine may bind to CaSR and/or T1R1/T1R3 heterodimers. However, we believe that ornithine mainly binds to GPRC6A, as a specific inhibitor of this receptor almost completely abolished the enhanced response to umami substances, and our immunohistochemical study indicated that GPRC6A-expressing taste cells are distinct from CaSR-expressing taste cells (see Supplemental Fig. 3). We conducted essentially the same experiments using gamma-Glu-Val-Gly in Wistar rats (Yamamoto and Mizuta, Chem. Senses, 2022) and compared the results in the Discussion. The reviewer may have misunderstood the chorda tympani results: we added the same concentration (1 mM) used in the two-bottle preference test to MSG (Fig. 5-B). Fig. 5-A shows nerve responses to five concentrations of plain ornithine. In the revised manuscript, we will strive to provide more precise information reflecting the reviewer’s comments.