**Sodium Imbalance in Mice Results Primarily in Compensatory Gene Regulatory Responses in Kidney and Colon, but Not in Taste Tissue**

**Kristina Lossow 1,2, Wolfgang Meyerhof 1,3 and Maik Behrens 1,4,\***


Received: 12 March 2020; Accepted: 30 March 2020; Published: 3 April 2020

**Abstract:** Renal excretion and sodium appetite provide the basis for sodium homeostasis. In both the kidney and tongue, the epithelial sodium channel (ENaC) is involved in sodium uptake and sensing. The diuretic drug amiloride is known to block ENaC, producing a mild natriuresis. However, amiloride is further reported to induce salt appetite in rodents after prolonged exposure as well as bitter taste impressions in humans. To examine how dietary sodium content and amiloride impact on sodium appetite, mice were subjected to dietary salt and amiloride intervention and subsequently analyzed for ENaC expression and taste reactivity. We observed substantial changes of ENaC expression in the colon and kidney confirming the role of these tissues for sodium homeostasis, whereas effects on lingual ENaC expression and taste preferences were negligible. In comparison, prolonged exposure to amiloride-containing drinking water affected β- and αENaC expression in fungiform and posterior taste papillae, respectively, next to changes in salt taste. However, amiloride did not only change salt taste sensation but also perception of sucrose, glutamate, and citric acid, which might be explained by the fact that amiloride itself activates bitter taste receptors in mice. Accordingly, exposure to amiloride generally affects taste impression and should be evaluated with care.

**Keywords:** epithelial sodium channel; sodium homeostasis; amiloride; salt deprivation; short-term preference test

#### **1. Introduction**

Sodium is the main cation in the extracellular fluid and the primary determinant of osmolarity, crucial for many biological processes [1]. Accordingly, levels of sodium are tightly controlled through a precise balance of sodium intake and excretion. The latter is primarily realized via the kidney, which plays a major role in volume, electrolyte, and blood pressure regulation [2,3]. To move sodium across the apical plasma membrane, the distal tubules and collecting duct of the kidney utilize the epithelial sodium channel (ENaC). In times of sodium deficits, aldosterone, a mineralocorticoid hormone produced in the adrenal glands [4], promotes the translocation of ENaC from cytoplasmic compartments to the apical plasma membrane in the renal collecting system [5] triggering sodium reabsorption. In comparison, sodium absorption in the intestine is primarily realized by the sodium/hydrogen exchanger rather than through the ENaC, which is limited to epithelial cells of the distal colon and rectum [6,7]. However, after

proctocolectomy, ENaC was verified in the distal part of the small intestine, indicating the importance of ENaC-mediated reabsorption of salt and water in the intestine [8]. Inhibition of ENaC is achieved by amiloride [9–12], first described by Cragoe in 1967 [13]. Amiloride lowers systemic blood pressure by preventing absorption of sodium and increasing its excretion along with water (natriuresis) [14–16].

Additionally, sodium homeostasis is maintained by ingestive behavior. Sodium appetite is the instinctive drive to seek salty substances or beverages for consumption [17] stimulated by sodium deficiency, hypovolemia, or mineralocorticoids. Accordingly, sodium-depleted rats ingest high sodium chloride solutions even at concentrations they would normally reject [18–21]. Already decades ago, studies revealed that amiloride not only affects the kidney but also acts as a potent blocker of salt taste in rodents [22–25]. Applied to the tongue before or during sodium stimulation, amiloride reduces sodium-evoked responses in the chorda tympani nerve innervating the fungiform papillae on the frontal tongue as well as the palate in the oral cavity [9,10,26,27]; vice versa, sodium deprivation or aldosterone application results in an increased number of amiloride-sensitive taste receptor cells in fungiform papillae [28].

Conclusively, ENaC has been detected in lingual epithelia and taste buds [9,10,23,29–33]. Fully functional ENaC is believed to be composed of three homologous subunits (α, β, and γ) arranged with a 1:1:1 stoichiometry [34,35]. In 2010, Chandrashekar and colleagues demonstrated the involvement of the α-subunit of ENaC in salt attraction and sodium taste responses by a tissue-specific knock-out model [32]. However, a recent gene-targeted approach, using fluorescent marker proteins under the control of the *Scnn1a* and *Scnn1b* gene loci, encoding α- and βENaC, respectively, concluded that the assumed αβγ-subunit composition of ENaC seems highly unlikely in taste tissue, as ENaC subunits were distributed in different taste bud cells under adequate sodium conditions [33].

Accordingly, the cellular and molecular composition of the "salt taste receptor" is still quite controversial with many unanswered questions. Furthermore, studies regarding the expression levels of ENaC subunits in gustatory tissues after dietary sodium restriction are rare and inconclusive [28,36]. To explore how sodium ingestion as well as short- and longtime exposure to amiloride impact on sodium appetite, we utilized a gene-targeted animal model with modified *Scnn1a* and *Scnn1b* loci, which were subjected to dietary and amiloride interventions, followed by taste reactivity testing (short-term preference test) and expression analysis of potential ENaC subunits.

#### **2. Materials and Methods**

#### *2.1. Animal Experiments*

All animal experiments were approved by and conducted following the national guidelines of the Ministry of Environment, Health and Consumer Protection of the federal state of Brandenburg (14513 Teltow, Germany; 2347-02-2017), and institutional guidelines of the German Institute of Human Nutrition Potsdam-Rehbruecke (14558 Nuthetal, Germany; T-01-17, T-02-17). Mice were housed in polycarbonate cages and kept under constant conditions (12 h light/dark cycles with light beginning at 6:00 am; 22 ◦C room temperature; 55% humidity). The animals received food and water *ad libitum* (for details see feeding experiments).

#### *2.2. Feeding Experiment and Amiloride Intervention*

In this study, either wild-type or homozygous double gene-targeted Scnn1a*IRES-GFP*/Scnn1b*IRES-tdRFP* mice (abbreviated Scnn1++/++ and Scnn1aa/bb, respectively, throughout) with modified *Scnn1a* and *Scnn1b* loci were used. These carried modified alleles allowing the synthesis of green (GFP) and red (tdRFP) fluorescent proteins in cells expressing α- and βENaC subunits, respectively. Details of modification/gene-targeting were described previously [33].

Before the experiments, Scnn1++/++ and Scnn1aa/bb animals had free access to water and standard chow diet (V1534, Ssniff, Soest, Germany; sodium content of 0.24%). From the 7th week of life on, the animals continued to have free access to water, while the chow was replaced either by a sodium-adequate (equal sodium content as within the chow, however 4.8 above nutrient requirements of laboratory animals) [37], sodium-deficient or high salt diet, with 0.21% (0.5% NaCl; E15430-04, Ssniff, Soest, Germany), <0.03% (E15430-24, Ssniff, Soest, Germany), and 1.71% sodium (4% NaCl; E15431-34, Ssniff, Soest, Germany), respectively. Body weight, food, and water intake were monitored weekly from weaning onward (21 days). Blood pressure was recorded prior to diet change/experimental session (6th week) and after 4 weeks of intervention (10th week) using Power Lab and Chart 5 software (ADInstruments Ltd., Oxford, UK). Urine samples were collected during the day (6 am to 5 pm) and overnight (5 pm to 6 am) at week 10, while mice were individually housed in metabolic cages (TechniPlast, Buguggiate, Italy). Urinary sodium content was determined in triplicate with a LAQUA twin system (Horiba Scientific for Na+, Darmstadt, Germany). Until then, all animals went through the same experimental procedure differing only in the sodium content of the diet. To test the effect of the consumed sodium on RNA expression levels and the distribution of fluorescent proteins, a randomly selected experimental set of mice were killed at the 11th week of life and subjected either to taste bud isolation (*n* = 4–6 per diet and genotype) or perfusion (*n* = 3 per diet and genotype). In a different experimental set, taste responses to different taste solutions were assessed by a short-term preference test. The remaining mice further continued with the dietary intervention for additional 4 weeks (*n* = 10–11 per diet and genotype) with defined access to water and food (for details see short-term preference test). In the third experimental setup, taste responses to different taste solutions after pre-treatment with amiloride were examined. In this experiment, mice fed a sodium-adequate diet either received access to amiloride-containing (300 μM) or amiloride-free water for 36 h [38] prior to short-term preference tests (*n* = 10–16 per intervention and genotype). Amiloride treatment was restricted to a maximum of 3 times for each animal, with a recovery phase of 36 h in-between (with amiloride-free water). After the final intervention/third amiloride treatment, animals were subjected either to taste bud isolation (*n* = 4 per intervention and genotype) or perfusion (*n* = 3 per intervention and genotype).

#### *2.3. Short-term Preference Tests*

Short-term preference tests were performed using the DavisRig system (MS-160, DiLog Instruments, Tallahassee, FL, USA), permitting several taste stimuli to be presented in a brief trial within a single test session. During these experiments, mice were singly housed with defined access to water and food. Mice were initially trained for 3 days after 18 h water restriction, using water as test stimulus to get used to the shutter system. To monitor attractive taste responses, animals were restricted for 22.5 h with limited access to food (1 g of the corresponding diet) and water (2.5 mL). For aversive taste stimuli, mice were water restricted over a period of 22.5 h with free access to food, as reported earlier [39]. Mice performed preference tests at the beginning of the light phase, followed by 1 h with free access to water and food. Test sessions were restricted to 2 or 5 consecutive days for attractive or aversive taste stimuli, respectively. To control for small individual differences in lick rate, number of licks to each taste stimulus was divided by the average number of licks to water alone for each mouse and day. A lick ratio of 1.0 reveals that licks to taste stimulus equals licks to water. In contrast, a lick ratio close to 0 indicates only a few licks of taste stimulus relative to licks for water, and a lick ratio > 1.0 signifies more licks of the taste stimulus relative to that for water. In case of apparent decreasing motivation of the animals, for example, reduced water licks under aversive test conditions or diminished licks for sucrose and sucralose under attractive restriction conditions, we excluded the entire subsequent test session. Accordingly, sometimes only the first 15 instead of 20 min were taken into consideration, adjusted day by day, and according to the motivational behavior of each mouse.

Taste solutions for testing were prepared daily with reagent grade chemicals and distilled water, sucrose (10–1000 mM, Merck, Darmstadt, Germany), monopotassium glutamate (MPG; 1–100 mM; Fluka, Oberhaching, Germany), denatonium benzoate (DB; 0.1–10 mM; Sigma, Taufkirchen, Germany), citric acid (CA; 1–100 mM; Roth, Karlsruhe, Germany), sodium chloride (NaCl; 10–1000 mM; Roth, Karlsruhe, Germany), inosine monophosphate (IMP; 1 mM; Sigma, Taufkirchen, Germany), and amiloride (0.1 mM; Sigma, Taufkirchen, Germany). Solutions were presented at room temperature. The sequence of stimulus presentation was randomized with varying taste solutions and concentrations (no ascending or descending presentation of a single taste solution) every day to minimize systematic order and contrast effects.

#### *2.4. Tissue Preparation*

Scnn1++/++ and Scnn1aa/bb mice were either anesthetized by intraperitoneal injection of 150 mg/kg body weight pentobarbital (Narcoren from Merial, Hallbergmoos, Germany) followed by transcardial perfusion with phosphate-buffered saline (PBS; 0.01 M Na2HPO4, 1.764 mM KH2PO4, 2.683 mM KCl, 0.1369 M NaCl, pH 7.4) and 4% paraformaldehyde (PFA) to gain tissue for cryosections (14 μm) and subsequent immunohistochemistry or with isoflurane (Cp-pharma, Burgsdorf, Germany) to gain tissues for RNA extractions as reported earlier [33].

#### *2.5. Immunohistochemistry*

Immunohistochemistry was performed as described before [33]. Primary antibodies included TrpM5 (1:5000; [40]) and aromatic L-amino acid decarboxylase (AADC, also known as DOPA decarboxylase, 1:500; GTX30448, GeneTex, Irvine, CA, USA).

Co-localization analysis in fungiform papillae was based on confocal (Leica TCS SP8) z-stack scans through tissue sections, of which a single plane was used for evaluation. Mean fluorescence intensity was evaluated in the operating mode of TCS SP8 LAS X software by defining a region of interest (ROI), namely one taste bud, in the digitized graph. Collected data reflect results from tissue sections of at least 3 mice per intervention.

#### *2.6. RNA Isolation and qRT-PCR*

Taste cells, lingual epithelium, kidney, and distal colon were subjected to RNA isolation and cDNA synthesis as previously reported [33]. Afterwards, 12.5 ng/well cDNA served as a template for quantitative RT-PCR using TaqMan Gene Expression Master Mix (Applied Biosystems by ThermoFisher Scientific, Foster City, CA, USA) and the corresponding probes (αENaC, Mm00803386\_m1; βENaC, Mm00441215\_m1; γENaC, Mm00441228\_m1; Eef2, Mm01171435\_gH; all ABI Applied Biosystems by ThermoFisher Scientific, Foster City, CA, USA; β-actin probe 5 FAM-TTGAGACCTTCAACACCCCAGCCA-3 TAM, β-actin for 5 -TACGACCAGAGGCATACAG-3 , β-actin rev 5 -GCCAACCGTGAAAAGATGAC-3 ; Eurofins MWG Operon, Martinsried, Germany) in a final volume of 10 μL. After initial 10 min denaturation at 95 ◦C, PCR was carried out for 40 cycles with 95 ◦C for 15 s and 60 ◦C for 60 s using Quant Studio 12K Flex Real-Time PCR System (Applied Biosystems by ThermoFisher Scientific, Foster City, CA, USA). Relative expression was determined based on normalization to β-actin and eukaryotic translation elongation factor 2 (Eef2) mRNA levels. As amplification efficiencies of all used probes were quite similar, cycle threshold (CT) values were averaged from triplicates and differences between CT values of ENaC and the housekeeping genes were calculated as ΔCT for normalization and finally expressed as 2−<sup>Δ</sup>CT. Collected data reflect results from isolated tissue of at least 4 mice.

#### *2.7. Functional Expression Analysis*

Functional expression analysis of bitter taste receptor cDNAs has been reported in detail earlier [39]. For the screening for amiloride-responsive bitter taste receptors, the compound has been applied to human embryonal kidney cells transiently expressing mouse (Tas2rs) and human bitter taste receptors (TAS2Rs) at a concentration of 3 mM. Subsequently, hits were verified by dose-concentration analyses, with concentrations ranging between 0.0001 and 3 mM. Latter data reflect results from 3 individual experiments.

#### *2.8. Statistics*

Data are given as means ± standard deviation (STABW; tables) or standard error (STE; figures). Statistical reliability of the observed results in the feeding study was determined by UNIANOVA and post-hoc analysis using Bonferroni's multiple comparison test (SPSS, SPSS 16.0, IBM, Chigago, IL, USA). For quantitative expression analysis, significant differences between 3 diets were determined by UNIANOVA (SPSS, SPSS 16.0, IBM, Chicago, IL, USA), followed by a comparison of individual pairs of means using Bonferroni's post-hoc test, whereas comparison between 2 intervention groups was conducted by using Student's unpaired *t*-test. Differences were considered to be statistically significant if *p* ≤ 0.05.

#### **3. Results**

#### *3.1. Feeding Experiment*

To investigate if diets with varying sodium content impact on the expression of ENaC, we fed Scnn1++/++ and Scnn1aa/bb mice with low, adequate, and high salt diets over a period of 4 weeks (from 7th to 10th week of age). We monitored body weight, food, and water intake from weaning onward. As expected, body weight constantly increased during the experiment independent of diet and genotype (*p* < 0.001; Figure 1A). Mice consumed equal amounts of food, ranging from 1.3 g after weaning to 3.7 g, with a mean intake of 2.5 g per day, irrespective of the sodium content of the diet (Figure 1B). In contrast, water intake changed after the diet switched (Figure 1C). When fed a high salt diet, mice increased their daily water intake by about 78% in comparison to animals fed with a diet adequate in sodium. The water intake of mice on low, adequate, and high salt diet ranged around 3.6 ± 0.5 mL, 3.6 ± 0.6 mL, and 6.5 ± 0.7 mL per day, respectively (*p* < 0.001). Furthermore, we observed significant differences in water intake based on genotype from the 5th week on, with significantly higher values in Scnn1aa/bb animals (Figure 1C).

In addition, we measured blood pressure prior and at the end of the dietary intervention (Figure 1D). Prior to diet change, no significant differences for mean systolic blood pressure were observed. After 3.5 weeks, animals that received the high salt diet showed slightly increased systolic blood pressure compared to animals that ate low or adequate salt diets, triggering significant diet X genotype effects (*p* = 0.012). Furthermore, urine was collected in metabolic cages at the end of the dietary intervention. Urinary volumes of mice fed the high salt diet were slightly higher, relative to the other groups. However, differences did not reach significance when considered for day, night, or total volume, due to high variations between the animals (Figure 1E). Considering mean urinary sodium content, significant variances were observed depending on diet (significant differences between all groups) and diet X genotype (*p* < 0.001), but not based on genotype itself. Thereby, no significant differences between groups fed low and sodium-adequate diet were recognized, whereas animals receiving the high salt diet showed significantly higher urinary sodium content in comparison to all other groups (Figure 1F).

#### *3.2. Expression Analysis*

In order to examine the impact of dietary salt on the expression of the ENaC subunits, we measured the transcription levels in the gustatory tissue, kidney, and distal colon of Scnn1aa/bb mice. Quantitative RT-PCR analyses of isolated fungiform and foliate/vallate taste buds and of non-gustatory lingual tissue showed no differences in relative expression levels of all 3 ENaC subunits after feeding a high or low salt diet in comparison to Scnn1aa/bb animals fed a control diet over a period of 4 weeks (Table 1). In kidney, αENaC expression was significantly reduced after consumption of a high salt diet, whereas β- and γENaC were not affected (Table 1). In the distal colon, intervention with a low salt diet resulted in significantly higher expression levels of α- and βENaC in comparison to Scnn1aa/bb animals fed with a standard or high salt diet. For γENaC, such significant variances were only detected between

Scnn1aa/bb animals fed a low and high salt diet, with higher expression levels for animals who received a low salt diet (Table 1).

**Figure 1.** Physiological parameters of Scnn1++/++ and Scnn1aa/bb mice during dietary monitoring. (**A**) Body weight, (**B**) food and (**C**) water intake, (**D**) blood pressure, (**E**) urinary volume, and (**F**) sodium excretion of Scnn1++/++ (*n* = 11) and Scnn1aa/bb mice (*n* = 19–20) fed with low salt, adequate, and high salt diet over a period of 4 weeks (7th to 10th week, time point of diet change is indicated by underline); after initial 3 weeks of sodium adequate chow diet (4th to 6th week) after weening (3rd week). Statistical differences between 2 bars at a specific time point are indicated by different letters based on UNIANOVA and post-hoc analysis using Bonferroni´s multiple comparison test.

**Table 1.** Relative expression of epithelial sodium channel (ENaC) subunits in Scnn1aa/bb mice after dietary intervention. Based on quantitative RT-PCR, the relative expression levels of ENaC subunits normalized to the housekeeping genes β-actin and eEf2 in isolated taste buds and non-gustatory tissue were determined. Data represent the means of 6 Scnn1aa/bb mice, fed with sodium-adequate, low, or high salt diet over a period of 4 weeks. Statistical testing is based on UNIANOVA and post-hoc analysis using Bonferroni´s multiple comparison test. *p*-Values rely on comparison of all groups with significant differences indicated in bold; individual differences between the different diets are additionally indicated as the following: \* statistical significance between sodium-adequate and high salt diet; # statistical significance between sodium-adequate and low salt diet; \$ statistical significance between low and high salt diet based on Student´s *t*-test.


Furthermore, no differences were noted regarding expression of the fluorescent reporter proteins in fungiform taste buds of Scnn1aa/bb animals. Consumption of either the low or high salt diet did not affect the location of GFP and tdRFP fluorescence, nor the fluorescence intensity. Accordingly, GFP always co-localized with Type III cell marker AADC, whereas tdRFP was not co-expressed with Type II or Type III cell markers TrpM5 or AADC, respectively (Figure 2).

However, ENaC expression of Scnn1aa/bb in comparison to Scnn1++/++ animals consuming an adequate salt diet revealed overall higher expression levels (Table S1). Whereas non-gustatory tissue differences in the expression levels of ENaC subunits did not reach statistical significance, prominent differences were recognized in lingual taste papillae affecting all ENaC subunits. In kidney, genotype primarily affected α- and βENaC subunits, whereas in the distal colon only βENaC expression was significantly increased in Scnn1aa/bb animals.

**Figure 2.** Expression of fluorescent proteins in fungiform papillae after dietary intervention. Fungiform papillae sections of Scnn1aa/bb animals expressing GFP (synthesis of green) and tdRFP (synthesis of red) fluorescence in αENaC- and βENaC-expressing cells, respectively, were stained for Type II (TrpM5) and Type III (AADC) taste cell markers after dietary intervention. Therefore, animals received either an adequate, low, or high salt diet over a period of 4 weeks. Independently of the consumed diet, GFP and tdRFP fluorescence showed no co-localization in taste papillae. Whereas GFP-positive cells always co-expressed AADC, tdRFP-positive cells revealed no overlap with the cell markers TrpM5 or AADC, visualized by immunofluorescence (white). Scale bars apply to all images.

#### *3.3. Short-term Preference Tests*

In order to determine if the sodium content of diets affects taste preferences for NaCl or other tastants, despite the lack of changes in gustatory ENaC expression, we performed a dietary intervention study. A group of 10 to 11 Scnn1++/++ and Scnn1aa/bb mice was fed an adequate, low, or high salt diet for another 4 weeks (total intervention of 8 weeks, from week 7–15). During this time, short-term preference tests were performed under conditions allowing the analysis of taste responses to attractive (Figure 3A–D) and aversive (Figure 3E–H) taste stimuli. For this procedure, it was mandatory to deprive animals of water. Even though the mice that received the high salt diet drank significantly more water under *ad libitum* conditions (Figure 1C), water restriction did not have an impact on the water lick rates during 5 s measuring periods. Accordingly, taste preference tests were carried out under the same conditions for all animals, independent of diet or genotype. Mean lick responses are shown in Figure 3.

Under conditions for testing attractive stimuli there was no diet X genotype effect for sucrose (Table S3). However, ANOVA revealed an effect for concentration with increasing number of licks/water at 30 mM sucrose for Scnn1aa/bb animals under the high salt regime (Figure 3A). For MPG+IMP, there were effects for concentration and diet X genotype (Figure 3B, Tables S2 and S3). The latter was observed at 1.0, 3.0, and 10 mM, with Scnn1aa/bb animals showing increased lick rates (Table S2). NaCl taste solutions were licked more at the low concentration of 30 mM by Scnn1aa/bb animals under the high salt regime and resulted in higher lick numbers at the high salt concentration of 300 mM by Scnn1aa/bb animals pretreated according to the low salt regime (Figure 3C, Tables S2 and S3). These effects were eliminated by amiloride treatment (Figure 3D, Tables S2 and S3). Control stimuli like IMP and denatonium benzoate presented only at a single concentration did not result in any diet X genotype effects (Figure S1A, Tables S2 and S3). In comparison to that, the control stimuli amiloride and citric acid revealed such effects, with higher lick/water ratios in Scnn1aa/bb animals (Figure S1A, Tables S2 and S3). For amiloride, highest lick/water means were observed for Scnn1aa/bb animals fed with a high salt diet, which were even higher than for animals fed the low or adequate diet, indicating that in this case not only genotype but also diet has an effect.

Additionally, animals were further water-restricted for 22.5 h to perform short-term preference tests for aversive stimuli (Figure 3E–H, Tables S2 and S3). Here, all taste stimuli were affected by concentration and diet X genotype (Table S3). For denatonium benzoate, Scnn1++/++ mice showed higher mean licks for denatonium benzoate at 0.1 to 1.0 mM than other diet X genotype constellations (Figure 3E). Scnn1++/++ animals further tended to show higher lick to water ratios for citric acid at 1 and 10 mM (exception: low salt fed animals at 1 mM; Figure 3F). In comparison to that, NaCl did not result in significant differences at 10 to 100 mM, whereas Scnn1aa/bb and Scnn1++/++ mice fed with a low salt diet and Scnn1aa/bb animals fed with an adequate diet showed higher lick ratios than Scnn1++/++ animals fed with an adequate diet or animals fed with a high salt diet (both genotypes; Figure 3G). Independent of diet, we observed changes in lick ratios among Scnn1++/++ and Scnn1aa/bb animals for 1 mM MPG+IMP, 100–1000 mM NaCl (attractive regime) as well as for 10 mM and 100 mM citric acid, 300–1000 mM NaCl, and 1000 mM NaCl+amiloride (aversive regime), respectively, when testing diet adequate in sodium content (*p* ≤ 0.05, Student's *t*-test).

**Figure 3.** Taste response curves of Scnn1++/++ and Scnn1aa/bb mice after dietary intervention. After 4 weeks fed with sodium-adequate, low, or high salt diet, Scnn1++/++ and Scnn1aa/bb mice were subjected to short-term preference tests using an automated gustometer. To do so, animals were either restricted for 22.5 h with access to 2.0 mL water and 1 g food (attractive restriction conditions, (**A**–**D**)) or water-deprived for 22.5 h (aversive restriction conditions, (**E**–**H**)). Taste solutions and concentrations were presented randomly. Each data point represents a mean ± standard error (SE) of 5 s presentations from the 10 to 11 animals tested. Statistical testing was based on UNIANOVA and post-hoc analysis using Bonferroni´s multiple comparison test. Significant differences over all groups in line drawings were indicated by asterisk(s) with \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.

#### *3.4. Amiloride Intervention*

As reported in a recent study, application of 300 μM amiloride over a period of 36 h induces robust salt appetite [38]. To investigate whether amiloride affects only the preference for sodium or also ENaC expression, Scnn1++/++ and Scnn1aa/bb mice were subjected to short-term preference tests as well as to taste bud and tissue preparations after receiving amiloride for 36 h next to a sodium-adjusted diet. According to the latter and subsequent expression analysis, free access to amiloride-containing water did not have an impact on the relative expression values for α-, β- or γENaC subunits in non-gustatory tissue and kidney, whereas in fungiform and foliate/vallate papillae, β- and αENaC, respectively, were induced after amiloride treatment (Table 2). However, no impact of amiloride intervention on GFP and tdRFP fluorescence was recognized in fungiform papillae with regard to localization and apparent intensity (Figure 4). According to expression analysis, all 3 ENaC subunits of the distal colon were affected by amiloride intervention, with significantly higher relative expression levels after access to amiloride for 36 h (Table 2).

**Table 2.** Relative expression of ENaC subunits in Scnn1aa/bb mice after amiloride intervention. Based on quantitative RT-PCR the relative expression levels of ENaC subunits normalized to the housekeeping genes β-actin and eEf2 in isolated taste buds and non-gustatory tissue were determined. Data represent means of 6 Scnn1aa/bb mice, with access to a sodium-adequate diet and water or 300 μM amiloride-containing drinking solution for 36 h prior to tissue isolation. Statistical testing was based on Student´s *t*-test. Differences were considered to be significant if *p* < 0.05, as indicated in bold.


In the short-term preference test, 3 concentrations of sucrose, MPG+IMP, NaCl, and NaCl+amiloride representing attractive taste stimuli were tested (Figure 5, Tables S4 and S5). The main differences were due to amiloride treatment. However, few additional effects of genotype were recognized (Tables S4 and S5). Accordingly, at least medium and high concentrations of sucrose and MPG+IMP resulted in significantly fewer licks after amiloride intervention (Figure 5A,B), whereas NaCl presentations led to increased licks/water ratios (Figure 5C,D). Licks of water did not show significant changes upon amiloride treatment. Aversive control stimuli (denatonium benzoate and citric acid) were only checked at a single concentration, whereas denatonium benzoate did not result in any differences between the groups and lick responses for citric acid were reduced after amiloride treatment (Figure 5E, Table S4).

**Figure 4.** Expression of fluorescent proteins in taste papillae after amiloride intervention. Fungiform papillae sections of Scnn1aa/bb animals expressing GFP (green) and tdRFP (red) fluorescence in αENaCand βENaC-expressing cells, respectively, were stained for Type II (TrpM5) and Type III (AADC) taste cell markers after amiloride intervention. Therefore, animals received adequate salt diet without or with 300 μM amiloride-containing drinking water prior to sacrifice. Independent of intervention, GFP and tdRFP fluorescence showed no co-localization in taste papillae. Whereas GFP-positive cells always co-expressed AADC, tdRFP-positive cells revealed no overlap with the cell markers TrpM5 or AADC, visualized by immunofluorescence (white). Scale bar applies to all images.

In addition, amiloride treatment did not only change perceived intensity of taste solutions but also increased motivational behavior of the animals. Accordingly, amiloride-treated mice initiated more trials/completed a larger percentage of trials during the 20 min test sessions than did mice that received only water. A trial began with the opening of the shutter and ended 5 s after the mouse made its first lick on the drinking spout (see Section 2). In each session, trials were set to a maximum of 50. Whereas most of the time, animals performed about 20–30 trials per day (e.g., "water-treated" mice, Figure 5 or after dietary intervention, Figure 3), amiloride-treated animals performed about 40–50 trials per session (Figure S2). Despite considerable individual variations for all animals independent of intervention, amiloride-treated animals showed reduced latency, or the time taken to initiate the first lick of a trial, reaching statistical significance for at least one concentration of a presented stimulus. Only for concentrated control stimuli (denatonium benzoate and citric acid) was no or a converse situation

recognized, however, it did not reach statistical significance (Figure S2E). Accordingly, the overall performance of amiloride-treated mice was changed.

**Figure 5.** Taste responses of Scnn1++/++ and Scnn1aa/bb mice after access to amiloride-containing water. Scnn1++/++ and Scnn1aa/bb mice receiving a sodium-adequate diet had either access to 300 μM amiloride-containing water 13 h prior to restriction starting or received water without amiloride. The restriction phase lasted for 22.5 h with access to 2.0 mL water ± 300 μM amiloride and 1 g of food. Lick responses to different concentrated solutions of sucrose (**A**), monopotassium glutamate with inosine 5´monophosphate (MPG+IMP; **B**), sodium chloride (NaCl; **C**), NaCl with amiloride (NaCl+amiloride; **D**), or bitter and sour stimuli (**E**) were determined by an automated gustometer. Each data point represents a mean ± SE of 5 s presentations from 10 to 16 animals tested. Statistical was testing based on UNIANOVA and post-hoc analysis using Bonferroni´s multiple comparison test. Different letters indicate statistical significance.

#### *3.5. Amiloride Interaction with Bitter Taste Receptors*

Amiloride was reported to be tasteless to rats and mice at or below 100 μM [41–43], whereas humans perceive bitterness at concentrations above 100 μM [44–47]. If, however, amiloride would also cause bitter perception in mice, this could have an unspecific impact on salt intake in case of synchronous application of amiloride. In order to confirm that amiloride could (not) activate bitter taste receptors, we performed functional expression analyses with mouse and human bitter taste receptor constructs. Transient expression of 25 human and 34 mouse bitter taste receptors revealed that amiloride indeed activated 1 and 7 bitter taste receptors, respectively (Figure 6). The activation of mouse bitter taste receptor Tas2r121 was observed at concentrations of 0.01 mM and above (Figure 6A), whereas the human bitter taste receptors, TAS2R4, TAS2R7, TAS2R13, TAS2R38, TAS2R39, TAS2R43, and TAS2R46 revealed an activation by amiloride at ~100 times higher concentrations (Figure 6B).

**Figure 6.** Concentration-response relations of murine (**A**) and human (**B**) bitter taste receptor-expressing cells stimulated with increasing concentrations of amiloride calculated from calcium traces acquired by fluorometric imaging plate reader (FLIPR) recordings. Changes in fluorescence (Δ*F*/*F*) were plotted semi-logarithmically versus agonist concentrations.

#### **4. Discussion**

Modern society is characterized by high consumption of table salt [48–52], which increases risks of diseases such as stroke, left ventricular hypertrophy, renal stones, or osteoporosis [53–60]. Accordingly, it is of considerable interest to elucidate and understand the molecular and cellular basis of salt taste. In 2010 the relevance of the αENaC subunit in attractive salt taste in mice was confirmed by a conditional knock-out in taste bud cells, impairing amiloride-sensitive salt taste detection, while retaining normal responses to other taste qualities as well as high salt reception [32,61]. This is in agreement with the observation that salt taste is, at least in mice, partially affected by the diuretic drug amiloride, a well-known effector of ENaC in the kidney [9–12]. Based on functional expression experiments in *Xenopus* oocytes, formation of a fully functional ENaC depends on the simultaneous presence of α-, β-, and γ-subunits, even though the α-subunit alone is sufficient to induce weak sodium currents [62–66]. However, a recent study with gene-targeted mice, labeling α- and βENaC-expressing cells by fluorescent proteins, revealed almost no co-localization of the different subunits in taste papillae [33]. To investigate if table salt restriction or overconsumption affects ENaC expression, we used the same α- and βENaC knock-in animals for a dietary intervention study. Over a period of 4 weeks, animals received either an adequate, low, or high salt diet with free access to food and water. Variable sodium content of the diet did not result in any changes in averaged food intake (Figure 1B), supporting the assumption that the caloric need determines the amount of food that is ingested rather than the sodium content. From the time point of dietary change, animals receiving a high salt diet showed a drastic increase in water intake, whereas sodium depletion caused no alterations in water intake in comparison to animals fed

an adequate sodium diet (Figure 1C). Accordingly, only ingestion of the high salt diet provided a rapid osmotic stimulation of thirst, as stated before for rats [67,68]. While some reports observed that sodium depletion by low salt diet did not enhance dietary sodium ingestion [69,70], others reported an induction of sodium appetite [71]. Our data suggest the absence of compensatory sodium ingestion. As oral sodium consumption rapidly quenches sodium appetite [72,73], an altered hedonic valance of sodium depending on the body's needs is assumed [71,74,75]. To measure the 'liking' of taste solutions, short-term preference tests were performed (Figure 3). In order to test the wide range of behavioral responses, attractive and aversive restriction conditions were applied. Under conditions favoring attraction behavior, we observed a higher mean lick ratio at 300 mM NaCl in Scnn1aa/bb animals fed with adequate and low salt diet in comparison to the other groups, whereas lick ratios for NaCl in Scnn1aa/bb animals fed with a high salt diet gradually decreased with NaCl concentration (Figure 3C). In comparison, Scnn1++/++ mice were relatively indifferent to all NaCl concentrations in comparison to water and did not show concentration-dependent changes in their lick ratio. Under water-restricted (aversive) conditions Scnn1++/++ and Scnn1aa/bb animals fed with low salt diet as well as Scnn1aa/bb animals fed with sodium-adequate diet revealed less aversive behavior towards hypertonic saline solutions (300 and 1000 mM) than animals fed with high salt diet or Scnn1++/++ animals fed with sodium-adequate diet (Figure 3G). Accordingly, depending on the table salt content of the diet, mice became significantly more adept at avoiding high salt-containing solutions to satisfy the demand for an adequate sodium supply. This is in line with the observation that sodium depletion does neither alter perceived intensity nor quality, assuming that only the hedonic character of table salt is affected [76]. Indeed, sodium-depleted human subjects also displayed an increased preference for high salt diets [77]. Moreover, sodium-depleted rodents drink sodium-containing solutions even at concentrations they would normally reject [18]. Accordingly, sodium deficits seem to trigger an intake behavior towards concentrated sodium solutions.

Furthermore, preference for table salt was reduced by co-application of amiloride without remaining variations between the different diet X genotype constellations under attractive restriction conditions; fitting to the observation that exposure to amiloride diminishes licks to table salt [78,79]. The co-application of amiloride and NaCl under water-restricted conditions had only modest effects on NaCl avoidance (Figure 3H), supporting the view that amiloride-insensitive pathways mediate the detection of high sodium concentrations [80–83].

Conspicuously, next to the diet, expression levels of ENaC subunits also seem to affect NaCl taste. Mice lacking Engrailed-2, a transcription factor critical for neural development, showed an increase in the expression of αENaC subunits in lingual taste papillae, accompanied by increased taste responsivity to 300 mM NaCl and reduced avoidance of salt [84]. Knock-in of fluorescent proteins in the here used Scnn1aa/bb mice also resulted in higher mRNA expression levels of ENaC subunits in taste papillae, but not in non-gustatory lingual tissues (Table S1). These differences in gene expression are probably due to the endowment of both loci with bovine growth hormone polyadenylation signal (BGH) as part of the gene targeting strategy [33]. The presence of the BGH signal results in increased stability and by that mild overexpression of α- and βENaC mRNA [85,86], potentially resulting in the production of higher polypeptide levels from the recombinant locus and eventually accounting for differences between both genotypes (Tables S2 and S3). This is seen, for instance, in the case of the Scnn1aa/bb knock-in mice fed with a sodium-adequate diet, who showed higher lick/water ratios for NaCl (100 to 1000 mM) than Scnn1++/++ mice under attractive restriction conditions (Figure 3C). In comparison to these observations, Scnn1aa/bb animals fed with a low salt diet seem more susceptible to the exposure of high NaCl concentrations, resulting in higher lick ratios at 100, 300, and 1000 mM in comparison to corresponding Scnn1++/++ animals (Figure 3C). This effect was not seen if NaCl was accompanied by amiloride (Figure 3D). Moreover, it seems that Scnn1aa/bb knock-in mice overexpressing α- and βENaC in general are more sensitive to low MPG+IMP and less sensitive towards high concentrated salt and sour stimuli.

Compared to genotype, either feeding of low or high sodium-containing diets failed to significantly alter relative RNA expression levels of ENaC subunits in gustatory tissue (Table 1). These observations confirm earlier studies, reporting unchanged transcription levels for ENaC subunits in taste buds of sodium-deprived rodents compared with animals fed a control diet [28,36,87,88]. Additionally, no changes in fluorescent protein expression (GFP and tdRFP) were observed, neither regarding cellular localization nor intensity in fungiform papillae. Accordingly, GFP fluorescence was only recognized in Type III cells, whereas tdRFP fluorescence was restricted to non-Type II and -Type III cells (Figure 2). However, with regard to physiologically potentially more relevant tissues for sodium homeostasis, elevated ENaC expression was recognized in the distal colon of animals, which were fed a low salt diet (Table 1). These results confirm and extend earlier studies which reported that low salt diet or application of aldosterone resulted in increased transcription of β- and γENaC subunits in the colon, whereas αENaC subunit transcription remained unchanged [36,89–91]. Some groups further reported that sodium-depleted rats not only showed altered ENaC mRNA levels, but also higher αENaC protein levels in the colon in comparison to animals fed a high salt diet [89,90]. In comparison to that, mice fed a high salt diet showed significantly reduced αENaC expression levels in the kidney in comparison to animals that received a sodium-adequate diet (Table 1). Previously, low salt diet or application of aldosterone was observed to induce the expression of the αENaC subunit in the kidney, accompanied with unchanged expression for β- and γENaC subunits [5,92–96], which was also not observed in this study. Accordingly, compensatory effects for maintaining sodium homeostasis are realized via the colon and kidney rather than taste tissue with regard to adaptations in ENaC expression, indicating the pivotal role of these tissues in sodium reabsorption [97,98].

In addition to dietary intervention, salt appetite in mice was reported to be induced more robustly by exposure to 300 μM amiloride in drinking water over a period of 36 h, as reported recently [38]. Access to 300 μM amiloride-containing drinking water for 36 h resulted in significantly higher expression levels for β- (and nearly also for αENaC) and αENaC in fungiform and posterior lingual papillae, respectively (Table 2). Additionally, once more in the distal colon, all ENaC subunit transcription levels were significantly increased after amiloride treatment (Table 2). Furthermore, lick responses to different taste solutions were altered (Figure 5, Tables S4 and S5). We observed increased lick ratios for NaCl potentially based on a sodium imbalance due to blocked ENaC channels (Figure 5C,D) as well as reduced lick/water ratios for most of the tested concentrations of sucrose and MPG+IMP (Figure 5A,B), indicating perceptual changes and/or reduced attractiveness to the mice. At least for humans, amiloride was recognized to suppress sweet taste [99–101]. Cell culture experiments using a cell line stably expressing human sweet taste receptor revealed that in the presence of 3 mM amiloride responses to sweet tastants like sugars and artificial sweeteners were reduced [99–101]. As sweet and umami taste share molecular and hedonic similarities, comparable effects of long-term amiloride exposure to both taste qualities appear reasonable. Moreover, a reduced lick/water ratio was recognized for 100 mM citric acid (Figure 5E). Whether interactions of amiloride with αENaC, which is expressed in sour-mediating Type III cells, or acid-sensing ion channels (ASICs) might be responsible for this, requires further analyses. At least after short amiloride exposure, neural responses to citric or hydrochloric acid were not affected by amiloride in various species [99,102–104], including the mouse [105]. Otherwise, preliminary results (unpublished) showed that at concentrations above 100 μM amiloride resulted in reduced lick/water ratios in short-term preference tests, indicating that aversive taste perception is triggered. Previously, amiloride has been reported to taste bitter to humans at concentrations above 100 μM [44–47], whereas amiloride was reported to be tasteless to rats and mice at or below 100 μM [41–43]. Our functional expression analysis now identified Tas2r121 as a murine bitter taste receptor for amiloride (Figure 6). This new finding may also have an impact on the observed species differences concerning the amiloride sensitivity of salt taste [44,46,104]. Moreover, the finding that amiloride tastes bitter to mice suggests that amiloride treatment does not only block attraction to low sodium chloride concentrations, but also represents at the same time an aversive taste stimulus. This should be kept in mind when interpreting and planning such experiments.

In summary, this study showed that sodium depletion, feeding a hypertonic saline diet, and amiloride intervention impact taste liking and ENaC expression, with differences regarding subunits and organs. Thereby, colon and kidney seem to be of greater importance to compensate imbalanced sodium homeostasis than gustatory tissue based on the monitored ENaC expression levels. However, effects of genotype were also recognized. As Scnn1aa/bb animals showed higher levels of ENaC subunits (at least at cDNA level) than corresponding wild-type controls, changes of ENaC expression seem to have a prominent impact on taste liking even without dietary interventions. This needs to be addressed in detail in future studies. Additionally, we could confirm that the application of 300 μM amiloride in the drinking water is an efficient way to induce salt appetite. However, amiloride did not only alter taste sensation for salt but also for sucrose, MPG+IMP, and high concentrations of citric acid, indicating a more general influence of this drug and its bitter taste on taste perception.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/4/995/s1, Figure S1: Taste responses of Scnn1++/++ and Scnn1aa/bb mice to control stimuli after dietary intervention, Figure S2: Latency to initiate the first lick for different taste stimuli after access to amiloride-containing water, Table S1: Relative expression of ENaC subunits in Scnn1++/++ and Scnn1aa/bb mice, Table S2: Statistical significance of different factors on the short-term preference tests of Scnn1++/++ and Scnn1aa/bb animals after dietary intervention, Table S3: Statistical significance of different factors on the short-term preference tests of Scnn1++/++ and Scnn1aa/bb animals, Table S4: Statistical significance of different factors on the short-term preference tests of Scnn1++/++ and Scnn1aa/bb animals after amiloride intervention for 36 h, Table S5: Statistical significance of different factors on the short-term preference tests of Scnn1++/++ and Scnn1aa/bb animals after access to amiloride-containing drinking water for 36 h.

**Author Contributions:** K.L. contributed to study design, performed experiments, data analysis, and interpretation, drafted the manuscript, and prepared the final manuscript version; M.B. provided ideas and contributed to the design of the experiments, data interpretation, drafting the manuscript, and was involved in the preparation of the final manuscript version. W.M. provided conceptual ideas, contributed to the design of the experiments, data interpretation, and preparation of the final manuscript version. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the institutional budget of the German Institute of Human Nutrition provided by the federal ministry for education and research (BMBF) and the ministry for science, research, and culture (MWFK) of the State of Brandenburg, Germany.

**Acknowledgments:** The authors thank Stefanie Demgensky for excellent technical assistance and Ines Grüner for animal caretaking and experimental support. Additionally, the authors thank Karoline von Websky for providing Power Lab system for the analysis of systolic blood pressure as well as Veit Flockerzi for generously providing the anti-TrpM5 antiserum.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Kokumi Taste Active Peptides Modulate Salt and Umami Taste**

**Mee-Ra Rhyu 1,\*, Ah-Young Song 1, Eun-Young Kim 1, Hee-Jin Son 1, Yiseul Kim 1, Shobha Mummalaneni 2, Jie Qian 2, John R. Grider <sup>2</sup> and Vijay Lyall 2,\***


Received: 31 March 2020; Accepted: 22 April 2020; Published: 24 April 2020

**Abstract:** Kokumi taste substances exemplified by γ-glutamyl peptides and Maillard Peptides modulate salt and umami tastes. However, the underlying mechanism for their action has not been delineated. Here, we investigated the effects of a kokumi taste active and inactive peptide fraction (500–10,000 Da) isolated from mature (FIIm) and immature (FIIim) Ganjang, a typical Korean soy sauce, on salt and umami taste responses in humans and rodents. Only FIIm (0.1–1.0%) produced a biphasic effect in rat chorda tympani (CT) taste nerve responses to lingual stimulation with 100 mM NaCl + 5 μM benzamil, a specific epithelial Na<sup>+</sup> channel blocker. Both elevated temperature (42 ◦C) and FIIm produced synergistic effects on the NaCl + benzamil CT response. At 0.5% FIIm produced the maximum increase in rat CT response to NaCl + benzamil, and enhanced salt taste intensity in human subjects. At 2.5% FIIm enhanced rat CT response to glutamate that was equivalent to the enhancement observed with 1 mM IMP. In human subjects, 0.3% FIIm produced enhancement of umami taste. These results suggest that FIIm modulates amiloride-insensitive salt taste and umami taste at different concentration ranges in rats and humans.

**Keywords:** Korean soy sauce; kokumi; umami; salty; chorda tympani; amiloride-insensitive salt taste pathway

#### **1. Introduction**

Mammals use G-protein-coupled receptors (GPCRs) expressed in Type II taste receptor cells (TRCs) to detect bitter, sweet, and umami taste stimuli. While amiloride-sensitive salt taste is detected by Type 1 cells expressing epithelial Na<sup>+</sup> channels, Type II and Type III cells mediate amiloride-insensitive salt taste. Otopetrin-1 proton selective channel expressed in Type III TRCs detects sour taste stimuli [1–6]. Much progress has been made in the identification of taste receptors and the downstream signalling mechanisms involved in the transduction of salty, sour, sweet, bitter and umami taste qualities. However, psychophysical, neural, and cellular studies have long suggested that cell to cell interactions within taste buds and interactions between different taste receptors enhance or suppress taste responses [7,8]. The synergism between monosodium glutamate (MSG) and 5'-ribonucleotides, a distinct characteristic of umami taste, is an example of a binary taste interaction between agonists [9,10]. Additionally, umami peptides modulate bitterness by interfering with ligand binding to the human bitter taste receptor TAS2R16 [11]. Interactions between non-tastants and tastants can also modulate taste intensity. SE-1, a sweet receptor positive allosteric modulator, binds to the sweet receptor without activating it, but does so in a manner that causes the orthogonal ligands to bind with higher affinity [12,13].

Kokumi taste has the characteristics of enhancing continuity, thickness, and mouthfeel, and was first observed in an aqueous extract of garlic in an umami solution. Kokumi produces its effect despite minimally eliciting any taste on its own [14]. Sulfur-containing compounds and their γ-glutamyl peptides, including γ-Glu-Cys-Gly (GSH) were suggested to be kokumi-active substances [14–17]. Because GSH was identified as an endogenous modulator of the calcium-sensing receptor, which participates in calcium homeostasis in the body [18], identification of GSH as an active component suggests the involvement of calcium-sensing receptor in kokumi perception [19]. Subsequent sensory analyses using various extracellular calcium-sensing receptor agonists have shown that kokumi did have a taste-enhancing effect on sweet, salty, and umami taste [19]. Not only does the γ-glutamyl peptide elicit kokumi taste, but the Maillard [20] reacted peptides (MRPs), which are gradually formed by longer maturation of Korean soy sauce, Ganjang (JGN), have been suggested to play a role in the kokumi taste in humans [21]. JGN is generally stored at ambient conditions for a year, and for up to four years or more to attain full maturity. The taste characteristics of kokumi increase as the maturation progresses.

Salt taste is detected by at least two receptor-mediated pathways. One pathway is Na<sup>+</sup> specific and involves Na<sup>+</sup> influx into TRCs that express amiloride- and benzamil (Bz)-sensitive epithelial Na<sup>+</sup> channels (ENaCs) [22,23]. The second pathway is amiloride-insensitive and is cation nonselective, and does not discriminate between Na+, K<sup>+</sup> and NH4 <sup>+</sup> salts. The contribution of these two pathways varies in different taste receptive fields. Approximately 65% of TRCs in the fungiform taste buds exhibit functional ENaCs, 35% of TRCs in foliate taste buds are amiloride-sensitive, while TRCs in the circumvallate are completely amiloride-insensitive, and do not seem to express functional ENaCs [24]. Although at present the identity of the amiloride-insensitive Na<sup>+</sup> pathways in TRCs remains elusive, the amiloride- and Bz-insensitive salt taste receptors are the predominant transducers of salt taste in humans [25–27].

Investigations conducted using the Maillard reaction between peptides (1000–5000 Da) isolated from soy protein hydrolysate and xylose (Xyl-MRPs) have been known to enhance umami, continuity, and mouthfeel in umami solution, support the notion that MRPs are another class of kokumi substances [28]. Interestingly, Xyl-MRPs not only modulate umami taste, but also modulate salt taste. The effect of Xyl-MRPs on salt taste is observed at much lower concentrations than those that increase the umami taste [27]. Over a range of concentrations, Xyl-MRPs [27,29] reversibly enhanced the Bz-insensitive NaCl chorda tympani (CT) taste nerve response in rodents, whereas, at high concentrations, they inhibited the Bz-insensitive NaCl CT response. The effect of Xyl-MRPs on the Bz-insensitive NaCl CT responses were transient receptor potential vanilloid 1 (TRPV1)-dependent. In human sensory evaluation, at low salt concentrations, galacturonic acid MRPs (GalA-MRPs) [27] enhanced human salt taste perception. These data suggest that, in both rodents and humans, MRPs induce changes in amiloride-insensitive salt taste and umami taste.

In this paper, we investigated the effects of a naturally occurring MRPs fraction (500–10,000 Da, FII) isolated from mature (FIIm; 4-year old) and immature (FIIim; 1-year old) JGN on salty and umami taste responses in rodents and human subjects. Effects of FIIm and FIIim were investigated on the Bz-insensitive NaCl CT responses and their interactions with TRPV1 modulators, and glutamate CT responses in rats. Effects of FIIm and FIIim were investigated on behavioral responses to NaCl in C57BL/6 mice, and on the sensory evaluation of salty and umami tastes in human subjects. Our results demonstrate that FIIm produces concentration-dependent biphasic effects on amiloride-insensitive neural and behavioral responses to NaCl in rodents. Above the concentrations that modulate salty taste, FIIm enhanced CT responses to glutamate. In human subjects, FIIm produced concentration-dependent biphasic effects on salt taste perception and at higher concentrations enhanced umami taste. These results suggest that FIIm modulates salty and umami taste in rodents and humans via similar mechanisms.

#### **2. Materials and Methods**

#### *2.1. Isolation of FIIm and FIIim from JGN*

FII fraction containing MRPs of molecular weight (MW) ranging between 500 and 10,000 Da was isolated from immature (FIIim; 1-year old) and mature (FIIm; 4-year old) JGN with an ultra-filtration unit (Model 840, Amicon Inc., Beverly MA, USA) using YM-10 (MW cutoff 10,000 Da) and YC-05 (MW cutoff 500 Da) membranes (Millipore Co., Bedford, MA, USA) at 2–4 ◦C under N2 pressure. Each fraction was lyophilized and stored in a desiccated freezer at −20 ◦C until use. FIIm was further separated using YM5, YM3 or YM1 Millipore membranes that had a cut off MW of 5000, 3000 and 1000 Da, respectively. These fractions were: FIIma (MW 500–1000 Da), FIImb (MW 1000–3000 Da), FIImc (MW 3000–5000 Da) and FIImd (MW 5000–10,000 Da). FIIm and FIIim are the unfractionated MRPs and FIIm(a-d) are the sub-fractions of different molecular weight. Successive column chromatography was performed with FIIm to obtain aromatic, basic, acidic, and neutral conjugated peptide fractions using activated charcoal (60 cm long and 4.0 cm I.D.; Junsei Chemical Co. Ltd., Tokyo, Japan), cation-exchanger (60 cm long and 3.0 cm I.D.; Amberlite IRC-50), and anion-exchanger (60 cm long and 3.0 cm I.D.; Amberlite IRA 400, both from Sigma Co. Ltd., St. Louis, MO, USA) [30,31].

#### *2.2. CT Taste Nerve Recordings*

In contrast to glossopharyngeal nerve response to NaCl, the predominant NaCl CT response in rodents is amiloride sensitive. However, a significant part of the NaCl CT response is Bz- and amiloride insensitive across the concentration-response range of NaCl [32]. The identity of the amiloride-insensitive receptor presently at best remains elusive in the circumvallate taste receptive field. Our previous studies suggest that in the anterior tongue the amiloride-insensitive pathway is a non-selective cation channel that is sensitive to resiniferatoxin (RTX), N-(3-methoxyphenyl)-4-chlorocinnamide, SB-366791 (SB), capsazepine, iodo-RTX, and temperature [33,34]. We have previously investigated the effect of various salt taste modulators on the Bz-insensitive NaCl CT response using both rats and mice [27,29,32–36]. To compare the results of the effects of FIIm on neural responses to NaCl with previously published results, we monitored Bz-insensitive NaCl CT response in rats.

Animals were housed in the Virginia Commonwealth University (VCU) animal facility in accordance with institutional guidelines. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC #AD20116). Female Sprague-Dawley rats (150–200 gm) were anesthetized by intraperitoneal injection of pentobarbital (60 mg/Kg) and supplemental pentobarbital (20 mg/Kg) was administered as necessary to maintain surgical anesthesia. The animal's corneal reflex and toe-pinch reflex were used to monitor the depth of surgical anesthesia. Body temperatures were maintained at 37 ◦C with a Deltaphase Isothermal PAD (Model 39 DP: Braintree Scientific Inc. Braintree, MA, USA). The left CT nerve was exposed laterally as it exited the tympanic bulla and placed onto a 32G platinum/iridium wire electrode. CT responses were recorded and analyzed as described previously [27,29,32–36].

The composition of rinse and NaCl stimulating solutions is shown in Table 1. CT responses in rats were monitored while the anterior lingual surface was stimulated first with the rinse solution (R) and then with salt solutions containing 0.1–0.5% FIIim, FIIm and FIIm sub-fractions (FIIm(a–d)). The pH of the rinse solution and the salt solutions was adjusted to 6.1. In some experiments Bz (5 μM) was added to salt solutions to block Na<sup>+</sup> entry into TRCs through apical epithelial Bz-sensitive ENaCs. CT responses were also recorded at 23 ◦C and 42 ◦C. In additional experiments we tested the effect of FIIm on the CT response to MSG and MSG + 5'-inosine monophosphate (IMP), a specific modulator of umami taste [37]. CT responses to MSG were monitored in the presence of Bz to eliminate the contribution of Na<sup>+</sup> to the glutamate CT response [33] and SB (1 μM), a TRPV1 blocker [38]. In CT experiments the tonic (steady-state) part of the NaCl CT response or glutamate CT response was quantified and normalized to CT responses to 0.3M NH4Cl. The normalized data were reported as the mean (M) ± SEM of the number of animals (*n*). Student's t-test was employed to analyze the differences

between sets of data. Since we are comparing the normalized CT responses to NaCl + Bz before and after FIIim, FIIm or FIIm sub-fractions in the same CT preparation, paired t-test was used to evaluate statistical significance.


**Table 1.** Taste stimuli used for CT experiments.

4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) was used to buffer the pH of rinse and salt stimuli at pH 6.1. Bz (benzamil); RTX (resiniferatoxin); SB (SB-366791, N-(3-methoxyphenyl)-4-chlorocinnamide). All compounds were obtained from Sigma-Aldrich.

#### *2.3. Behavior Studies in Mice*

Rats have a high preference for NaCl even in the presence of Bz [29]. Because of already high background NaCl preference, small increases in NaCl preference are difficult to evaluate in rats. In contrast, mice demonstrate a more moderate preference for NaCl and small shifts in the preference curve are easily detected. Therefore, mice were used for behavioral studies. Behavioral studies were performed in WT (C57BL/6J) mice (30–40 gm) using standard two bottle/48 h tests [39]. Both males and females were used. The care and use of the mice followed the institutional and national guidelines, and the protocol was approved by the committee on the Ethics of Animal Experiments of the Korea Food Research Institute (Permit Number: KFRI-M-12028). Mice (63–70 days of age) were housed in separated cages and were maintained on a standard laboratory chow (Pico-Lab Rodent Diet 20–5053, PMI Feeds) and water *ad libitum*. The air-conditioned animal room was maintained at 22 ± 2 ◦C, with relative humidity of 59 ± 1% and a 12 h light/dark cycle (light period, 07:00–19:00 h). Each mouse was tested at approximately the same time of day. Before the start of the experiment mice were given two bottles with water for 2 weeks. The experiment was started when mice were accustomed to drinking equally from 2 bottles. Mice were given a choice between two bottles, one containing water and the other a test solution in the following order: water, 30 mM NaCl, 80 mM NaCl, 100 mM NaCl, 120 mM NaCl, 150 mM NaCl, 200 mM NaCl and 300 mM NaCl. We also performed behavioral studies when both water and the NaCl solutions contained 10 μM amiloride. In some experiments, mice were given a choice between water and 100 mM NaCl or between water + 10 μM amiloride and 100 mM NaCl + 10 μM amiloride containing varying concentrations (0.1, 0.25, 0.5, 0.75, and 1.0%) of FIIm. For each 48 h period the mass of water versus the mass of the test solution consumed by each mouse/g BW was measured. The preference ratio for a taste stimulus was calculated as the mass of the test solution consumed/48 h/g BW divided by the mass of the total fluid intake (mass water + mass of the test solution)/48 h/g BW). The bottles containing water or the test solution were switched from left to right every day. The data were analyzed using one-sample t-tests against 0.5, a reference value meaning indifference of the test solution with respect to the control solution.

#### *2.4. Human Sensory Evaluation*

All human sensory evaluation protocols were approved by the Public Institutional Review Board Designated by Ministry of Health and Welfare, South Korea. The ethic approval code is P01-202004-23-004. Each participant signed a consent form to participate in salt taste sensory evaluations. To maintain a subject's confidentiality, the personal data was coded and the taste data were analyzed off line. Previously trained panelists (men and women, ages between 25 to 37 years) with no history of basic taste disorders were recruited. The panelists washed their mouth after tasting each samples. The data was analyzed by one-way ANOVA to compare between-group differences.

#### 2.4.1. Salt Sensory Evaluation

Panelists were trained to recognize salt taste intensity with reference to 0.2%, 0.35%, 0.5%, and 0.7% NaCl solution representing a value of 2.5, 5.0, 8.5, and 15.0, respectively, using a 15-point intensity scale [40]. To evaluate the effect of FIIim and FIIm on salt taste, FIIim or FIIm (0–0.01%) dissolved in 0.2% NaCl solution was presented to the panelists and the salt taste intensity was evaluated with reference to 0.2% NaCl (intensity scale value = 2.5; R1), and 0.35% NaCl (intensity scale value = 5.0; R2), respectively.

#### 2.4.2. Umami Sensory Evaluation

According to the manufacturer's instructions, Japanese fish soup base, Hondashi (0.04 g) was dissolved in 100 ml water. The 0.04% Hondashi fish soup base was used as a control and was given an intensity of 5 on a 10-point intensity scale. FIIm at 0.003%, 0.01%, 0.03% and 0.3% was dissolved in 0.04% Hondashi Fish soup base and their effect was evaluated on umami taste by the same trained panelists (*n* = 6). As a control, FIIm was dissolved in water at 0.003%, 0.01%, 0.03% and 0.3%. These concentrations of FIIm were evaluated for umami taste by the same panelists (*n* = 6).

#### **3. Results and Discussion**

#### *3.1. E*ff*ect of FIIm and FIIim on the Bz-insensitive NaCl CT Response*

As shown in a representative CT trace (Figure 1A), adding increasing concentrations of FIIm to 100 mM NaCl + 5 μM Bz (NaCl + Bz) solution (Table 1) initially produced an increase in both phasic and tonic NaCl CT response of between 0.1% and 0.5%. Above 0.5% FIIm the magnitudes of the phasic and tonic CT responses were less than their respective maximum values. In the presence of 1% FIIm, the tonic CT response decreased below the NaCl + Bz CT response in the absence of FIIm (Figure 1A). The variation of the normalized mean tonic NaCl + Bz CT response plotted as a function of the log of FIIm or FIIim concentrations (%) are summarized in Figure 1B. FIIm produced a biphasic dose-response relationship for both the phasic (data not shown) and tonic (Figure 1B; •) NaCl + Bz CT response. The maximum increase in the mean normalized tonic CT response occurred at 0.5% of FIIm, an 88% increase relative to NaCl + Bz tonic CT response in the absence of FIIm. At 1% FIIm, the tonic NaCl + Bz CT response was significantly less than the tonic CT response with NaCl + Bz alone (*p* = 0.0466; *n* = 3). Stimulating the tongue with the rinse solution (R) containing varying concentrations of FIIm elicited only transient (phasic) CT responses that were concentration-independent and were indistinguishable from the mechanical rinse artifact (data not shown). These results indicate that, at the concentrations used in these experiments, FIIm, by itself, is not a gustatory stimulus in the fungiform taste receptive

field and only modulate the CT response in the presence of salt (NaCl + Bz). In contrast, FIIim did not produce any changes in either the phasic (data not shown) or the tonic CT response between 0.1% and 1% (Figure 1B; -). The Xyl-MRPs, GalA-MRPs, glucosamine-MRPs, and fructose-MRPs also produced biphasic effects on the Bz-insensitive NaCl CT response with maximum increase at 0.25%, 0.25%, 0.50%, 0.75% and 1%, respectively [27]. This suggests that both MRPs naturally generated during longer maturation and synthesized in vitro have a common property of producing biphasic effects on the Bz-insensitive NaCl CT response. The potency of MRPs depends upon the reacted sugar moiety. However, at present it is not known which sugar moieties are conjugated to the peptides comprising FIIm. FIIm is a mixture of MRPs of varying molecular weights, charge and affinity for their putative salt taste receptor(s). In comparison, 0.27% GalA-MRPs enhanced the Bz-insensitive NaCl CT response by 101% [27], suggesting naturally occurring FIIm produces effects on the NaCl + Bz CT response that are comparable to those produced by the GalA-MRPs.

**Figure 1.** Effect of FIIim and FIIm on the benzamil (Bz)-insensitive NaCl chorda tympani (CT) response. (**A**) Shows a representative trace in which the CT responses were monitored while the rat tongue was first superfused with a rinse solution (R) and then with a stimulating solution containing 100 mM NaCl + 5 μM Bz + FIIm (0–1%) maintained at room temperature. The arrows represent the time periods when the rat tongue was superfused with R and the stimulating solutions. The data were normalized to the tonic response obtained with 0.3 M NH4Cl. (**B**) Shows the mean normalized tonic NaCl CT responses in different sets of 3 rats each while their tongues were first stimulated with R and then with NaCl + Bz solutions containing 0–1% of the FIIm (•) or FIIim (-) expressed in log units. The values are M ± SEM of 3 rats.

#### *3.2. E*ff*ect of SB and FIIm on the Bz-insensitive NaCl CT Response*

In our previous studies, Bz-insensitive NaCl CT responses in rodents were inhibited by TRPV1 blockers. In addition, Bz-insensitive NaCl CT responses were not observed in TRPV1 knockout

mice [33]. Accordingly, in the next series of experiments we tested if FIIm effects on salt responses were also sensitive to SB, a TRPV1 blocker. Because 0.4% and 0.5% FIIm give almost equivalent CT responses (Figure 1B), we used 0.4% FIIm in these experiments. In mixtures containing NaCl + Bz + SB, the constitutive NaCl + Bz tonic CT response was inhibited to the rinse baseline level (Figure 2A,C). Subsequently, stimulating the rat tongue with solutions containing NaCl + Bz + SB + 0.4% FIIm significantly inhibited the CT nerve response relative to NaCl + Bz + 0.4% FIIm (Figure 2A,C; \*\* *p* = 0.0001; *n* = 3). These results suggest that both the constitutive amiloride- and Bz-insensitive CT response and the subsequent FIIm-induced increase in the CT response are SB-sensitive.

**Figure 2.** Effect of resiniferatoxin (RTX), SB-377791 (SB), FIIm and temperature on the benzamil (Bz)-insensitive NaCl chorda tympani (CT) response. (**A**) Shows a representative CT trace obtained while the rat tongue was first stimulated with rinse solution (R) and then with NaCl, NaCl + Bz, NaCl + Bz + 0.4% FIIm, NaCl + Bz + 0.25 μM RTX, NaCl + Bz + 0.4% FIIm + 0.25 μM RTX, NaCl + Bz + 1 μM SB and NaCl + Bz +SB + 0.4% FIIm maintained at room temperature. The data were normalized to the tonic response obtained with 0.3 M NH4Cl. The arrows represent the time periods when the rat tongues were superfused with R and the stimulating solutions. (**B**) Shows a representative CT trace obtained while the rat tongue was first stimulated with R at 23 ◦C (R23 ◦C) and then with NaCl + Bz (NaCl + Bz23 ◦C), NaCl + Bz + 0.4% FIIm at 23 ◦C (NaCl + Bz + FIIm23 ◦C), NaCl + Bz at 42 ◦C (NaCl + Bz42 ◦C) and NaCl + Bz + 0.4% FIIm at 42 ◦C (NaCl + Bz + FIIm42 ◦C). The trace also shows the CT response in the presence of NaCl + Bz + SB and NaCl + Bz + SB + 0.4% FIIm maintained at 23 ◦C. The data were normalized to the tonic response obtained with 0.3 M NH4Cl. The arrows represent the time periods when the rat tongues were superfused with R and the stimulating solutions. (**C**) Shows the M ± SEM normalized rat tonic NaCl + Bz CT responses at 23 ◦C and 42 ◦C in the absence and presence of 0.4% FIIm. All unpaired comparisons were made with respect to the normalized value of the tonic CT response to NaCl + Bz at 23 ◦C. \* *p* = 0.0038; \*\* *p* = 0.0001; *n* = 3).

#### *3.3. E*ff*ect of RTX and FIIm on the NaCl* + *Bz CT Response*

Consistent with previous studies [33,35], at room temperature (23 ◦C), RTX (0.25 μM) enhanced the rat NaCl + Bz CT response relative to NaCl + Bz (Figure 2A,C; \* *p* = 0.0001; *n* = 3). When the tongue was stimulated with NaCl + Bz solutions containing both RTX (0.25 μM) and FIIm (0.4%), no further increase in the magnitude of the Bz-insensitive NaCl CT response was observed relative to NaCl + Bz + RTX (Figure 2C). These results suggest that RTX and FIIm target the same amiloride-insensitive pathway(s). The Bz-insensitive NaCl taste responses are regulated by several intracellular signaling mediators. A decrease in taste cell Ca2+, activation of protein kinase C, and inhibition of calcineurin enhanced the magnitudes of the Bz-insensitive NaCl CT responses in the presence of RTX, and either minimized or completely eliminated the decrease in the CT response at RTX concentrations >1 μM. In contrast, increasing taste cell Ca2<sup>+</sup> inhibited the Bz-insensitive NaCl CT response in the presence of RTX [41]. An increase in taste cell phosphatidylinositol 4,5-bisphosphate inhibited the control NaCl + Bz CT response and decreased its sensitivity to RTX. Alternately, a decrease in phosphatidylinositol 4,5-bisphosphate enhanced the control NaCl + Bz CT response, increased its sensitivity to RTX stimulation, and inhibited the desensitization of the CT response at RTX concentrations >1 μM [42]. It is likely that Bz-insensitive NaCl CT responses in the presence of FIIm are also regulated by the above intracellular modulators and are responsible for their biphasic effects on the NaCl CT response.

#### *3.4. E*ff*ect of Elevated Temperature and FIIm on the NaCl* + *Bz CT Response*

In our previous studies, Bz-insensitive NaCl CT responses in rodents were temperature dependent. In addition, temperature and modulators of the Bz-insensitive NaCl CT response produced additive effects on CT response [26,33,36]. Accordingly, we next tested the effect of elevating the temperature from 23 ◦C to 42 ◦C on the CT response to NaCl + Bz and NaCl + Bz + 0.4% FIIm. As shown in a representative CT recording, elevating the temperature from 23 ◦C to 42 ◦C increased the magnitude of the tonic NaCl + Bz CT response relative to 23 ◦C (Figure 2B). FIIm (0.4%) further increased the CT response at 23 ◦C and 42 ◦C (Figure 2B). The mean tonic NaCl + Bz CT response at 23 ◦C (Figure 2C) was significantly enhanced by increasing the temperature to 42 ◦C (\* *p* = 0.0039) and by the addition of 0.4% FIIm (Figure 2C; \*\* *p* = 0.0001; *n* = 3). These results show that elevated temperature and FIIm produce additive effects on the amiloride-insensitive NaCl CT response.

#### *3.5. E*ff*ect of FIIm sub-fractions of Di*ff*erent Molecular Weights (FIIm(a-d)) on the NaCl* + *Bz CT Response*

FIIm was further separated into four sub-fractions of varying molecular weights: FIIma (500–1000 Da), FIImb (1000–3000 Da), FIImc (3000–5000 Da) and FIImd (5000–10,000 Da). As shown in representative CT recordings in Figs. 3A and 3B, the relationship between varying concentrations of FIIma and FIImc and the magnitude of NaCl + Bz CT response was shifted to the right on the concentration axis relative to FIIm (Figure 1A). The relationships between varying concentrations of FIIma, FIImb, FIImc and FIImd and the corresponding mean normalized tonic NaCl + Bz CT response are plotted in Figure 3C. The results show that for all sub-fractions FIIm(a–d), the relationship between their concentrations and the magnitude of tonic NaCl + Bz CT response is shifted to the right on the concentration axis relative to FIIm. FIIma produced the maximum increase in the NaCl + Bz tonic CT response at a concentration between 1.5 and 2.5% (Figure 3C; ¡). This concentration is significantly higher than the concentration at which FIIm produced the maximum increase in the NaCl + Bz CT response (0.5%; Figure 1B). These results suggest that FIIm fraction is composed of MRPs of varying molecular weights that differ in their affinity and potency in modulating the putative amiloride-insensitive salt taste pathway(s).

**Figure 3.** Effects of FIIm sub-fractions (FIIm(a-d)) on the benzamil (Bz)-insensitive NaCl chorda tympani (CT) response. Representative CT responses showing the effect of adding varying concentrations of FIIm sub-fractions FIIma (500–1000 Da) (**A**) and FIImc (1000–3000 Da) (**B**) on the rat CT responses to NaCl + Bz. The arrows represent the time period when the tongue was superfused with the rinse and stimulating solutions. In each rat the data were normalized to the tonic response obtained with 0.3M NH4Cl. (**C**) Shows the mean normalized tonic NaCl CT responses in different sets of 3 rats each while their tongues were first stimulated with R and then with NaCl + Bz solutions containing 0–1% of the four FIIm sub-fractions in log units. The values are M ± SEM of 3 rats in each group. In each case the data were fitted to Equation (4).

#### *3.6. E*ff*ect of FIIm sub-fractions (Neutral, Acidic, Basic and Aromatic) on the NaCl* + *Bz CT Response*

FIIm was further separated into neutral, acidic, basic and aromatic sub-fractions. Since the relationships between varying concentrations of the neutral, acidic and basic fractions and the magnitude of the tonic NaCl + Bz CT response were very similar in individual rats, the data from these three fractions were combined and are plotted in Figure 4A (-). In all three fractions, the relationship between their concentrations and the magnitude of the mean normalized tonic NaCl CT response was shifted to the right on the concentration axis relative to FIIm (Figure 4A; •). In contrast, the aromatic fraction produced a biphasic response in the NaCl + Bz CT response with a very sharp-peak at 0.75% (Figure 4A; -). These results further suggest that FIIm is composed of neutral, acidic, basic and aromatic MRPs that show varying degrees of potency and affinity for modulating the putative amiloride-insensitive salt taste pathway(s). It is interesting to note that relative to control (NaCl + Bz), 0.5% FIIm (Figure 1A) produced an equivalent maximum increase in the tonic NaCl + Bz CT response as 1 μM RTX (Figure 4B).

**Figure 4.** Effects of aromatic, neutral, acidic and basic FIIm sub-fractions on the benzamil-insensitive NaCl chorda tympani (CT) response. (**A**) Shows the relationship between varying FIIm sub-fraction concentrations expressed in log units and the mean normalized tonic NaCl CT response from 3 rats in each group for FIIm (•), aromatic (-) and combined neutral, acidic and basic maillard reacted peptides (-). (**B**) Shows the relationship between resiniferatoxin (RTX) concentrations expressed in log units and the mean normalized tonic NaCl CT responses from 3 rats (•). The values are M ± SEM of 3 rats in each group.

We also recorded FIIm-induced changes in the Bz-insensitive NaCl CT response in wild type (WT; C57BL/6J) and homozygous TRPV1 knockout mice (B6. 129S4-Trpv1tmijul; Jackson Laboratory, Bar Harbor, ME). Consistent with our earlier study with MRPs [27], FIIm produced a similar biphasic response on the Bz-insensitive NaCl CT response in WT mice. In TRPV1 KO mice, FIIm (0.4%) did not induce CT response above the rinse baseline (data not shown). This is akin to our results in rats. SB inhibited the basal Bz-insensitive NaCl CT response. In the continuous presence of SB, FIIm produced a significantly smaller increase in the Bz-insensitive NaCl CT response relative to the absence of SB (Figure 2). These results indicate that FIIm produces similar effects on rats and mice.

#### *3.7. E*ff*ect of Calcitonin Gene Related Peptide (CGRP) on NaCl CT Responses*

RTX activates and SB inhibits amiloride-insensitive NaCl CT responses (Figure 2). However, TRPV1 immunoreactivity was not found in TRCs [43–45]. We hypothesize that RTX and other modulators of TRPV1 alter Bz-insensitive NaCl CT responses indirectly, by releasing CGRP from trigeminal nerves [46]. The released CGRP then acts on its specific receptor (CGRPR) in TRCs to modulate Bz-insensitive NaCl CT responses [47].

Due to the concern that topical lingual application of CGRP, a large neuropeptide, may not be able to reach its receptor in TRCs, CGRP was administered by intraperitoneal injection. CT responses were monitored while the rat tongue was stimulated with 0.3M NH4Cl, 0.3 M NaCl and 0.1M NaCl before and after an i.p. injection of 23 μg/100 BW or 68 μg/100 g BW CGRP dissolved in 0.5 mL PBS. Following i.p. injection of 68 μg/100 g BW CGRP the NaCl CT response increased with time (data not shown). As shown in Figure 5A, 10 min post CGRP injection, the NaCl CT responses were almost two times greater than control (Figure 5B). CGRP also induced an increase in the CT response to 0.3M NaCl. However, an i.p. injection of 23 μg/100 BW CGRP did not induce any changes in rat NaCl CT response 10 min post CGRP injection (Figure 5B). These results indicate that CGRP effects on NaCl CT response are both time- and dose-dependent and are observed over a range of NaCl concentrations. These results suggest a possible interaction between the trigeminal and salt taste systems.

**Figure 5.** Effect of i.p. injection of calcitonin gene related peptide (CGRP) on NaCl chorda tympani (CT) response. (**A**) Shows a representative CT trace obtained while the rat tongue was first stimulated with rinse solution (R) and then with 0.3M NH4Cl, 0.3M NaCl and 0.1M NaCl before and after i.p. injection of CGRP (68 μg/100 g BW in PBS). In each rat the data were normalized to the tonic response obtained with 0.3M NH4Cl. The values are M ± SEM of 3 rats in each group. (**B**) Shows summary of the data from 3 rats in each group injected with either 23 or 68 μg CGRP/100 g BW. Values are M ± SEM of 3 rats. \* *p* = 0.017 (0.1M NaCl) and 0.009 (0.3M NaCl).

Using calcium imaging, a subset of acid responsive Type III mouse circumvallate TRCs were identified as the amiloride-insensitive salt responsive cells [2]. CGRPR has been suggested as the functional link to cellular transduction pathway for CGRP action on Type III TRCs. CGRP has been shown to increase [Ca2+] in Type III TRCs. This effect of CGRP was dependent upon phospholipase C activation and was prevented by U73122 [47]. In mouse taste buds, CGRP caused TRCs to secrete serotonin (5-HT), a presynaptic (Type III) cell transmitter. 5-HT seems to reduce taste evoked ATP secretion in Type II cells [47]. However, at present this information is lacking in the fungiform taste receptive field.

Here, we present new data that suggest that CGRP can modulate rat amiloride-insensitive NaCl CT responses (Figure 5). In a recent study [3] amiloride-insensitive Ca2<sup>+</sup> responses in mouse taste bud cells were localized to the apical tips of Type II, but not in Type III fungiform TRCs. It is suggested that, because anterior (fungiform) and posterior (circumvallate) taste fields differ functionally, in an earlier study [2] amiloride-insensitive NaCl responses may have been detected in only Type III circumvallate taste cells. Although the identity and location of the amiloride- and Bz-insensitive pathway(s) are ambiguous at present, CT recordings demonstrate that a component of the amiloride- and Bz-insensitive NaCl CT response at low NaCl concentrations (100 mM) is present in the anterior taste field that is modulated by RTX, FIIm, temperature, SB (Figures 1A and 2B) and voltage [33,34].

*N*-geranyl cyclopropylcarboxamide (NGCC), a modulator of the amiloride- and Bz-insensitive NaCl CT responses [48], activates hTRPV1 expressed in HEK293T cells [49]. In our preliminary studies, component of FIIm induced inward current in TRPV1-expressing cells in whole-cell patch-clamp recordings [50,51]. Currently studies are underway to demonstrate direct activation of the expressed umami taste receptor by FIIm. However, at present it is not clear if, like RTX, other modulators of the amiloride-insensitive pathway release CGRP from TRPV1 expressed in trigeminal neurons in a dose-dependent manner. In addition, it is also not known if, like other modulators of the amiloride-insensitive NaCl CT response, CGRP elicits a biphasic effect on rat NaCl CT responses. Taken together, our data suggest a possible linkage between the trigeminal system and amiloride-insensitive salt taste. It has recently been demonstrated that sour taste pathway works together with the somatosensory system to trigger aversive responses to acidic stimuli [5].

#### *3.8. Behavioral Studies with Mice*

Under control conditions, mice demonstrated a bell shaped NaCl preference curve with a significant preference for 100 mM NaCl (Figure 6A; -; \* *p* = 0.02; *n* = 10) and aversion for 300 mM NaCl (\*\*\*\* *p* = 0.0001) [39]. In the presence of 10 μM amiloride the NaCl preference curve was again biphasic but was shifted to the right on the NaCl concentration axis. In the presence of amiloride, mice showed a significant NaCl preference at 150 mM NaCl (Figure 6 A; •; \*\*\* *p* = 0.0024).

**Figure 6.** Effect of amiloride and FIIm on NaCl Preference in WT mice. (**A**) Shows NaCl Preference in WT mice when given a choice between H2O and varying concentrations of NaCl (3, 80, 100, 120, 150, 200 and 300 mM) in the absence (-) and presence of 10 μM amiloride (•). The values are presented as mean (M) ± SEM of *n*, where *n* = 7–10. \* *p* = 0.02; \*\* *p* = 0.0134; \*\*\* *p* = 0.0024; \*\*\*\* *p* = 0.0001. (**B**) Shows NaCl Preference in WT mice when given a choice between H2O and 100 mM NaCl (-) or H2O and 100 mM NaCl + 10 μM amiloride (•) containing increasing concentrations of FIIm (0.1 to 1%). \* *p* = 0.0086; \*\* *p* = 0.0018; \*\*\* *p* = 0.0001 (*n* = 10). Dotted line represents the indifference value.

As shown in Figure 6B, adding increasing concentrations of FIIm (0.1 to 1%) to 100 mM NaCl solutions in the absence and presence of 10 μM amiloride produced biphasic changes in NaCl preference, increasing it at 0.25% and lowering it at higher concentrations. Under control conditions, FIIm maximally enhanced the NaCl preference at 0.25% relative to NaCl alone (Figure 6B; -; \*\* *p* = 0.0001; *n* = 10). Above 0.25% FIIm NaCl preference was significantly less than its maximum value. In the presence of 10 μM amiloride the maximum increase in NaCl preference was observed at 0.5% FIIm (Figure 6B; •; \* *p* = 0.0086). Above 0.5% FIIm NaCl preference was significantly less than its maximum value. There was no change in NaCl preference when equivalent concentrations of the FIIim were added to the test solutions containing 100 mM NaCl or 100 mM NaCl + 10 μM amiloride (data not shown). These behavioral responses to NaCl are correlated with the biphasic effects of FIIm concentrations on the

amiloride- and Bz-insensitive NaCl CT responses (Figure 1). In this sense, FIIm mimics the effect of other modulators [27,33,34,36,48,52] of the amiloride- and Bz-insensitive NaCl CT responses.

#### *3.9. E*ff*ect of FIIm on Salt Taste in Human*

In human sensory evaluation, FIIm produced a biphasic effect on salt taste. FIIm increased salt taste intensity between 0.3 and 0.5%, but slightly decreased it above 0.5% (Figure 7). The maximum salt taste intensity in human subjects was detected at 0.5% FIIm (Figure 7; •). In contrast, FIIim had no significant effect on human salt taste perception (Figure 7; -). In our previous studies, GalA-MRPs, Xyl-MRPs [27] and NGCC [48], modulators of the amiloride- and Bz-insensitive NaCl CT responses in rodents, also produced biphasic effects on human salt taste intensity. Although functional ENaC channels are expressed in human fungiform TRCs [53], the amiloride- and Bz-insensitive salt taste receptors are the predominant transducers of salt taste in humans [25–27]. Thus, modulation of the amiloride-insensitive salt taste in humans via FIIm or other modulators may provide alternate ways to regulated human salt taste and perhaps salt intake.

**Figure 7.** Effect of FIIm and FIIim on human salt taste intensity. Shows the effect of varying concentrations (0.03 and 1.0%) of FIIim (-) and FIIm (•) expressed in log units on human salt taste intensity. R1 corresponds to the intensity (2.5) of 0.2% NaCl and R2 corresponds to the intensity (5.0) of 0.35% NaCl. FIIm showed a significant (\* *p* = 0.01) salt taste-enhancing activity at 0.003% and 0.005%. In contrast, no effect of FIIim was observed on human salt taste intensity over the concentration range between 0.03 and 1.0%.

#### *3.10. E*ff*ect of FIIm on the Rat CT Response to Glutamate*

Stimulating the rat tongue with 100 mM MSG + Bz + SB elicited a CT response and the CT response was enhanced in the presence of 1 mM IMP (Figure 8A). Glutamate CT response was also enhanced in the presence of 2.5% FIIm. The normalized tonic CT responses to glutamate in the absence and presence of IMP and FIIm are summarized in Figure 8B. FIIm at 2.5% enhanced the CT response to glutamate that was equivalent to the enhancement observed with 1 mM IMP. These results further suggest that unlike the Bz-insensitive NaCl CT response, the basal umami CT response and the subsequent FIIm induced enhancement of the umami CT response is SB-insensitive. In our previous study, Xyl-MRs also enhanced the CT response to glutamate at concentrations above which they modulated the NaCl + Bz CT responses. In contrast to Xyl-MRPs, at these concentrations GalA-MRPs or Glc-NH2-MRPs did not show effects on the glutamate CT response [27]. These results suggest that the umami enhancing effect of MRPs is dependent on the conjugated sugar(s). However, at present the identity of the specific sugar resides conjugated with the peptides comprising the FIIm is not known.

**Figure 8.** Effect of FIIm on the glutamate chorda tympani (CT) response and human umami taste sensory evaluation. (**A**) Shows a representative CT response in which the rat tongue was first rinsed with the rinse solution (R) and then with 100 mM MSG + 5 μM benzamil (Bz) + 1 μM SB-366791 (SB), MSG + Bz + SB +1 mM IMP, MSG + Bz + SB + 1% FIIm and MSG + Bz + SB + 2.5% FIIm. The arrows represent the time period when the tongue was superfused with the rinse and stimulating solutions. (**B**) Shows mean normalized tonic CT responses from 3 rats. In each rat the data were normalized to the tonic response obtained with 0.3 M NH4Cl. \* *p* = 0.001. (**C**) Shows the effect of adding increasing concentrations of FIIm (0.003 to 0.3%) to the 0.04% Fish Soup Base (open bars) or to H2O (filled bars). The values are presented as M ± SEM of n, where n represents the number of panel members tested. \* *p* = 0.01.

In our earlier study [33], RTX demonstrated a biphasic response on the rat Bz-insensitive NaCl CT response. At 1 μM, it maximally enhanced and at 10 μM, maximally inhibited the Bz-insensitive NaCl CT response. At 1 and 10 μM concentrations, RTX did not alter CT responses to 500 mM sucrose, 10 mM quinine and 10 mM HCl. These results tend to suggest that over the concentration range that alter the Bz- insensitive NaCl CT response, modulators of the amiloride-insensitive pathway may not alter sweet, bitter or sour taste. At present it is not known if FIIm concentrations that modulate salt responses also alter responses to other taste stimuli.

#### *3.11. E*ff*ect of FIIm on Umami Taste in Human*

In human subjects, adding 0.3% FIIm to umami soup base produced a significant increase in umami taste intensity (Figure 8C; open bars; *p* < 0.01; *n* = 9). Equivalent concentrations of FIIm added to water produced umami intensity ratings of < 1 (Figure 8C; filled bars). In contrast to a strong salt taste enhancing effect at 0.5% FIIm, lower concentrations (<0.3%) of the FIIm did not have a significant effect on umami taste intensity. Thus, depending upon the concentration, MRPs can be used either as salt taste or umami taste modifiers.

These results show that FIIm modulates both salt and umami taste in humans but at different concentration range. The differences in the sensitivity to FIIm between humans and mice are most likely due to the variations in the umami taste receptor protein [54,55]. This dual property of being able to modulate the Bz-insensitive NaCl response and the glutamate response at two different concentration ranges is not restricted to FIIm. We have recently shown that at different concentrations ranges, NGCC modulates Bz-insensitive salt taste responses and glutamate taste responses in humans and animal models [48].

#### **4. Conclusions**

In summary, a naturally occurring kokumi taste active peptide fraction (MW 500–10,000 Da) isolated from mature (FIIm; 4-year old) Ganjang, a typical Korean Soy Sauce, modulates the amiloride-, Bz-insensitive NaCl CT response in rodents in a biphasic manner. At low concentrations (0.1 to 0.5%) it enhanced and at higher concentrations (>0.5%) inhibited the Bz-insensitive NaCl CT response. FIIm effects on Bz-insensitive NaCl CT responses are TRPV1 dependent. FIIm may indirectly alter CT responses to NaCl via the release of CGRP from trigeminal fibers near the fungiform taste buds in the anterior taste field. This suggests a novel relationship between trigeminal system and salt taste perception. At concentrations >1%, FIIm enhanced the CT response to glutamate. In human sensory tests, FIIm increased the salt taste intensity between 0.3 and 0.5%, and the umami taste intensity at 0.3%. We conclude that, depending upon its concentration, FIIm modulates both salty and umami tastes in humans and rodents. The active component(s) and salt enhancing property of naturally occurring MRPs by longer maturation in food should be further investigated for a better understanding of the potential link between the compound and its beneficial effect in reducing salt intake in the human population.

**Author Contributions:** Conceptualization, M.-R.R. and V.L.; methodology, E.-Y.K., S.M.; validation, E.-Y.K.; formal analysis, A.-Y.S., H.-J.S., J.Q; investigation, A.-Y.S., H.-J.S., S.M., J.Q.; data curation, Y.K.; writing—original draft preparation, M.-R.R. and V.L.; writing—review and editing, M.-R.R. and V.L..; funding acquisition, M.-R.R., V.L. and J.R.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Korea Food Research Institute, grant numbers E069002 and E0131201, the National Research Foundation of Korea (NRF), grant number NRF2020R1A2C2004661, National Institute of Health, grant numbers DK34153 (J.R.G.), DC-005981 (V.L.), and DC-011569 (V.L.), VCU Wright Center for Clinical and Translational Research 2019 and VCU Health under the Value and Efficiency Teaching and Research program 2019 (V.L.). The APC was funded by NRF.

**Acknowledgments:** We gratefully acknowledge the assistance of MJ Kim of Korea Food Research Institute.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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