J. Biol. Macromol., 14(1), 43-57 (2014)

Article

Relationship between GAD/GABA and Sweet, Umami and Bitter , Those Received in Type II Buds

Kimiko Sasaki,1,2,* Kaori Hamano1 and Hiroshi Ueno1

1Lab. Appl. Microbiol. & Biochem., Nara Women's University, Nara 630-8506, JAPAN 2Dept. , Mimasaka University, Tsuyama 708-8511, JAPAN

Received February 25, 2014; Accepted March 3, 2014

Food extracts from spices and teas were examined for the taste altering ability on sweet, umami and bitter tastes by using the taste sensation test. The idea arose based upon the findings that 1) some food extracts from spices and teas act as taste enhancers and 2) the enhancing effect is directly proportional to glutamate decarboxylase 67 (GAD67) activity in vitro. Our results indicated that those extracts significantly activated or inhibited GAD67 enzyme activity were able to alter sweet, umami, and bitter tastes. However, there were no relationships between the taste potency of each of the examined extracts and the GAD67 relative activity ratio. While it is unlikely that the extracts have directly activated GAD67 activity and enhanced sweet, umami or bitter tastes, the results do not exclude the idea that GABA may participate in the taste cell-to-cell communications.

Key words: GAD, GABA, taste signal transduction

In Japan, several decades ago people used to Introduction have struggled to obtain enough food to Food supplies important nutrients and satisfy their needs, but nowadays they can energy to sustain life for humans. Human choose to eat from available in their eating behaviors vary depending on social daily lives. The purpose of eating circumstances and cultural environments. gradually has shifted from satisfying huger to concerning nutritional aspects as well as ______*To whom correspondence should be enjoyment of eating, and taste becomes a addressed: E-mail: [email protected] critical factor in eating. Abbreviations: GABA: γ-amino butyric acid; GAD, glutamate decarboxylase; PCA, Five basic tastes, sweet, sour, bitter, perchloric acid; PLP, pyridoxal 5'- salty, and umami, have been established (1). phosphate 44 Association of GAD/ GABA and tastes

Taste substances first interact with connections (4). It has not been clear that corresponding receptor proteins and the how taste signals from type II cells signals are transmitted to the taste nerve transmitted to the nerves. Moreover, the system. Those receptor proteins locate on role of the chemical mediators that may act the taste buds, which are classified into 4 cell on the taste signal transduction has not been types, I, II, III, and IV (2). Type II cells clarified. Recently, Nakamura et al. have express G-protein coupled receptors for shown that glutamate decarboxylase (GAD) sweet, bitter and umami tastes, and type III 67, one of the isoforms to synthesize - cells express ion-channel receptors for sour aminobutyric acid (GABA), is expressed in and salt tastes. Although taste signals the type III taste cells (5)(Fig.1). They also transmitted from receptors to taste nerves, found that GABA-gated chloride ion type II cells appears to have no synaptic channel, also known as GABAA receptor, is connection with gustatory nerves (3). expressed in the taste buds (5-7). Electron microscopic observation showed Nakamura's study has expanded our only type III cells having synaptic understanding of the role of type III cells,

Fig. 1 Schematic view for taste transduction route between type II and type III cells Sweet, umami and bitterness are received on type II cells. Salty and sourness are received on type III cells that have synaptic connections. GAD67 synthesizes GABA inside of type III cells. Sasaki, Hamano and Ueno 45

where GABA may play an active role in the fluctuation of GABA level could occur salt signal transduction. within the type III cells, it should influence GAD67 expressed in the type III cells is salty taste as well as other associated an active enzyme and produces GABA (5). activities. In fact, Hisaki and Ueno have Since GABA is a known in shown that extracts of spices and herbs CNS, it is reasonable to assume that GABA significantly altered GAD67 enzymatic plays some biological role(s) in the type III activity in vitro and this phenomenon cells. In this extent, two types of GABA coincided with the level of salty taste (6, 8). actions involving the taste signaling are Hence, it is in value to examine the proposed. One is an intracellular action of fluctuation of GABA level and its influence GABA in the sour taste signaling, and the on sour, umami and sweet tastes. other an extracellular action in sweet, bitter, The contrast effect has been known in and umami taste signaling. If any the food cookery field. A small amount of Table 1 Characteristics of food extracts added salt significantly enhances the A name and components of spices used in the and/or umami of foods. extraction of food ingredients Inhibition effect is also known that a small added amount of salt weakens bitterness. It is still a puzzle that how salt which acts on type III cells could influence sweetness, bitterness and umami whose taste information is received on type II cells. Since any signals traveling from type II to type III, or vice versa, have not been clearly understood, the contrast and/or inhibition effects on foods could not be explained at molecular level. If GABA could be identified as a messenger acting between type II and type III cells, the mechanism of the contrast and/or inhibition effects could be explained. In order to demonstrate the GABA role as the messenger between the two cell types, we present preliminary results of 1) how food extracts affect sweet, bitter or umami tastes and 2) whether or not those effects are related to GAD67 activity.

46 Association of GAD/ GABA and tastes

production. GAD reaction was initiated by Materials and Methods adding the appropriate amount of enzyme Extraction and preparation of food solution into final 1.0 mL assay solution to ingredients which 100 μL of the assay mixture Spices, herbs and tea leaves listed in containing 0.2 M L-glutamate and 2 mM Table 1 were obtained from Somatech pyridoxal 5'-phosphate (PLP) in 0.5 M Center, House Foods Co., and commercially HEPES buffer, pH 7.0 was added. The available sources. Most samples were assay solution was incubated at 37 °C for 1 provided as a dry form and were processed h. At the end of incubation period, 50 μL of into a small pieces. Ultrapure water was 60% perchloric acid (PCA) was added to added to the samples and placed in a terminate the reaction. As a blank, PCA refrigerator for overnight. Then, water solution was added to the assay solution soluble components were filtered using 110 immediately after the enzyme addition. mm diameter filter paper. Crushed food After the centrifugation, the supernatant was samples were typically mixed with 5 times collected, where an appropriate amount of weight volume of water and powdered the aliquots was placed into the vial and samples were mixed with 10 times weight injected to HPLC for GABA analysis as volume of water. The supernatants were described previously (8). Area of the collected and used as food extracts. GABA peak was compared to that of the standard GABA and the amount of GABA Preparation of GAD67 produced per incubation time was estimated. Recombinant rat GAD67 was Together with protein assay data obtained by expressed by growing E. coli strain Bradford method (BioRad), specific activity transformed with Rosetta-gamiB (DE3) of GAD67 was calculated as expressed pLysS vector in which GST-GAD67 mol/min/mg. Bovine serum albumin was encoding cDNA was cloned in this used as a reference protein for the protein laboratory. Purification of the recombinant assays. GAD67 protein from GST-GAD67 fusion protein was carried out by glutathione Evaluation of the effects of food extracts on affinity chromatography. GST-tag was GAD67 activity removed with thrombin treatment to give an Effects of food extracts on GAD67 active GAD67. activity was evaluated by carrying out the enzyme assay in the presence of an Evaluating GABA production appropriate amount of food extracts. The GAD67 enzyme assay was carried out amount of GABA produced was compared by quantitatively determining GABA with that obtained without the addition of the Sasaki, Hamano and Ueno 47

food extracts. Any increase or decrease in their effects on human taste sensation (9). GAD67 activity was referred to relative Basic tastes for sweet, umami and bitter activity ratio (%) where no change in were 3% sucrose, 0.08% MSG and 0.05% activity was expressed as zero and any caffeine, respectively (Table 2) (10-12). inhibition was expressed as a minus % Reagents used were commercial grade number. purchased from NacalaiTesque, Ltd. Each of the extract solutions was diluted with Taste sensory test ultrapure water to the appropriate Seventeen extracts obtained from the concentrations and placed in a paper cup. spices, herbs and teas were examined for Typically, about 20 ml was supplied for the

Fig.2 Effects of food extracts on GAD67 Activity GAD67 activity is plotted in the presence and absence of food extract. Increased or decreased activity is indicated as positive or negative number in %, respectively. Water was used in place of food extracts as control that gave 100%. 48 Association of GAD/ GABA and tastes

Table 2 Reagents used in sensory test

tests. Detailed test procedure is described 2. Take the first tasting solution into as follows: mouth trying to spread over the entire (1) Sensory test subjects were selected surface of the , and spit out or from a group of students ranged in age from swallow it. 19 to 21 at Mimasaka University where the 3. Rinse mouth with ultrapure water, tests were performed. They agreed with then, take the second tasting solution the purpose of this study. They were into mouth. volunteers and did not receive any special 4. Complete the sensory evaluation sheet training prior to the tests. The study was in three step scale, “strong”, “same” approved by the Mimasaka University and “weak” on any changes. Ethics Review Committee. (2) The sensory test was proceeded Results according to the guideline "Fact of Food Search of spices that affect the activity of Sensory Test to Measure the Taste" (13). GAD67 (3) Subjects were allowed to repeat the We have examined if spice, herb and tests if they were not fully confidence of the tea extracts influenced the activity of results. GAD67 by incubating with those ingredients (4) Tests were limited to three or four and measured any changes in GABA times in order to avoid fatigue of the subjects. production with and without the extracts (5) The test site was at the cooking (Fig. 2). peel, anise, cumin, and laboratory on campus, where room raised GABA production whereas temperature was kept at 23-24 °C. All tests rose rugosa, chamomile, lemongrass, oolong, were performed between 9:00 and 12:00 a.m. and oregano showed strong inhibition. (6) Test results were statistically processed Among the examined, a small number of the according to the two-sample preference test extracts activated GAD67 activity. The (14). level of GABA production varied among the (7) The subjects proceeded the sensory test extracts. GABA contamination in the protocol as follows: extracts was considered; however, it is 1. Rinsed mouth with ultrapure water Sasaki, Hamano and Ueno 49

Table. 3 Change in the sweet taste sensitivity by food extracts （n＝30） The subjects were evaluated three levels "strong", "same" and "weak" the strength of bitter taste after tasting the food ingredients. enhancing effect of sweet taste Relationship between activity of GAD67 and enhancing effect of sweet by Food Extracts. Enhancing effect of taste by the food extract is subtracted a ratio of panelists who evaluated that taste was weak from the rate of them who evaluated that taste was strong. Enhancing effect of sweet taste points = strong - weak /n (number of subjects)

unlikely occurred since endogenous GABA activity in vitro. was subtracted from GAD assay results by the control experiments. Hence, it is likely Sensory test that there is a direct interaction between We conducted a sensory test in which GAD67 protein and food ingredient(s). 30 female college students answered the Our results support the idea that spice or tea strength of sweet, umami and bitter tastes, components have an impact on GAD67 whereas the 17 extracts were tested due to 50 Association of GAD/ GABA and tastes

their affects on GAD67 activity. concentration for sucrose was shown to be 0.5%. The amounts of the extract were (1)Sweet taste determined based upon their effects on Sweet taste tests were carried out with GAD67 activity. Results are summarized 3% sucrose solution in the presence and in Table 3. Those who tasted the extracts of absence of the extracts. This concentration all 17 extracts answered that anise, poppy of sucrose was chosen according to the seed, celery, mace, lemongrass and oolong reference (10). The threshold tea enhanced sweet taste. On the other hand, Table. 4 Change in the umami taste sensitivity by food extracts （n＝29） The subjects were evaluated three levels "strong", "same" and "weak" the strength of bitter taste after tasting the food ingredients. enhancing effect of umami taste Relationship between activity of GAD67 and enhancing effect of umami by Food Extracts . Enhancing effect of taste by the food extract is subtracted a ratio of panelists who evaluated that taste was weak from the rate of them who evaluated that taste was strong. Enhancing effect of umami taste points = strong– weak /n (number of subjects) Sasaki, Hamano and Ueno 51

over 60% of the test subjects responded that concentration of 0.08% was used as an basil, chamomile, ginger and rosa rugosa umami compound. This concentration was weakened sweet taste. It is evident that chosen as it was above the threshold spices, herbs and teas could alter sweet taste. (0.03%) (10, 11). Subjects were asked to taste the umami solution in the absence and (2)Umami taste presence of an appropriate amount of each of L- solution at the extracts. The subjects marked “strong”

Table. 5 Change in the bitter taste sensitivity by food extracts （n＝29） The subjects were evaluated three levels "strong", "same" and "weak" the strength of bitter taste after tasting the food ingredients. enhancing effect of bitter taste Relationship between activity of GAD67 and enhancing effect of bitter by Food Extracts . Enhancing effect of taste by the food extract is subtracted a ratio of panelists who evaluated that taste was weak from the rate of them who evaluated that taste was strong. Enhancing effect of bitter taste points = strong - weak /n (number of subjects) 52 Association of GAD/ GABA and tastes

and “weak” when they sensed umami taste salty taste enhancement. They suggested being enhanced and diminished, that the presence of food extracts could respectively. Table 4 summarized the reduce salt intake and the reduction of salt results. Anise, cumin, lemongrass, oregano, intake could lead to the prevention of celery, mace and parsley significantly hypertension. Although detailed enhanced umami taste. Perilla and ginger mechanism has not been clarified, the study significantly inhibited umami taste. suggests that the observed salt enhancement Similar to sweet taste, the components of is probably due to the effects on salt signal spices and teas could have affected umami transduction pathway locating on the type III taste. taste buds. In this study, we have focused on the effects of spice, herb, and tea extracts (3)Bitter taste on sweet, umami, and bitter tastes. Caffeine concentration of 0.05% was Spices, herbs, and teas may have some chosen for the bitter taste tests. This unexplored activities besides on the taste concentration of caffeine was typical for sensations, possibly altering sweetness, regular coffee and tea (12). Chamomile, umami and bitterness. It has not been anise, paprika and celery enhanced the bitter rigorously investigated on how spices, herbs taste (Table 5). Interestingly, there were no and teas affect the tastes except few cases extracts that significantly reduced the bitter (16, 17). Recently, a salty taste signal taste. In addition, the dynamic range for the transduction study has reported that GAD67 effects on bitter taste was relatively narrow is involved via producing GABA in type III as compared with those for sweet and umami cells, and some spice extracts exhibit salt tastes. Similar to sweet and umami tastes, enhancing effects. This finding has the examined extracts showed various encouraged us to study the roles of spices in effects on bitter taste. relation with tastes on human subjects. We have measured the potency of taste for each of the extracts in the sensory test. Discussion The potency of taste was derived by Modern biological and nutritional subtracting the number of subjects who sciences have shown that food not only responded that the extract "weakened" the provides energy, but also its components taste from the number of subjects who play significant physiological roles on our reported that the same extract "enhanced”. health (15). Recently, Hisaki et al. reported An index of the enhancing effect of taste by that food extracts could be used as a salty the food extract was thus calculated based taste enhancer (6). In their study, for example, a small amount of anise exhibited Sasaki, Hamano and Ueno 53

upon the taste potency that was divided by the extracts are responsible for the alteration the total number of subjects (Tables 3 - 5). of the tastes, and what the molecular basis or The results indicate that almost all the mechanism for such actions. examined extracts did alter the sweet, In addition to the roles of spice and tea umami and bitter tastes. Some enhanced extracts on sweet, umami and bitter tastes as while some others reduced sensation of one described above, the relationship between or more of the three tastes. At the moment, the effect on GAD67 activity (Fig. 2) and it is not clear which chemical components in each of the examined taste sensations were

poppy seed ○ celery ○ lemongrass ○ 0.4 ○ ○○ ) mace oolong anise ○ paprika 0.2

perilla sweet taste(points sweet ○ Y = 0.3213x+ 13.956 yuzu peel

R2 = 0.1516 ○ 0 ○ cumin ○ oregano ○ parsley peppermint Enhancing effect on Enhancing effect -0.2 ○ rose rugosa ○ chamomile basil ○ ginger ○ ○ -0.4

-80 -60 -40 -20 0 20 40

GAD67 Activity (%)

Fig. 3 Relationship for the effect of 17 food extracts on sweet taste sensation and that on GAD67 activity X axis indicates effects on GAD67 activity and Y axis indicates effect on the sweet taste. Correlation between the effects is shown as a line (R2 = 0.1516). 54 Association of GAD/ GABA and tastes

analyzed (Fig. 3 - 5): them. The results indicate that any activation or inhibition of GAD67 activity Sweet Taste may not influence sweet taste. This may We examined whether or not the effects deny the possibility that any GABA of those food extracts on sweet taste have produced in the type III cells would some of correlation with GAD67 activity. influence the sweet taste signal transduction. When the sweet taste potency vs relative It has been reported that the degree of the activity for GAD67 are plotted (Fig. 3), there sweetness was unaffected by the GABA seems to be no direct correlation between presence in the food samples (18, 19). This

○ ○ cumin 0.6 lemongrass

○ oregano ○ 0.5 anise mace celery 0.4 ○ ○ parsley poppy seed ○ ○ 0.3 chamomile ○ y = 0.06764x+ 28.52665 0.2 R = 0.01445 ○ peppermint 2 rose rugosa ○ paprika 0.1 ○ ○ ○ Enhancing effect on umami taste (points) Enhancing effect oolong basil ○ yuzu peel ginger 0 ○ perilla ○

-80 -60 -40 -20 0 20 40 GAD67 Activity (%) Fig. 4 Relationship for the effect of 17 food extracts on umami taste sensation and that on GAD67 activity X axis indicates effects on GAD67 activity and Y axis indicates effect on the umami taste. Correlation between the effects is shown as a line (R2=0.01445). Sasaki, Hamano and Ueno 55

suggests that GAD67 and GABA possibly The effects of the extracts on GAD67 locating in the type III cells have no relative activity ratio were plotted against communication with the sweet taste those on umami taste potency. As a result, transduction that is received by the type II there was no correlation between the cells. GAD67 relative activity ratio and the umami taste potency (Fig. 4). It is of interests to Umami Taste note that the degree of umami taste was not We examined if the umami taste was affected when GABA alone was added (18, affected by GAD67 activity and/or GABA. 19). This suggests that GAD67 activity and

○ chamomile 0.4 paprika ○ anise Y= 0.04409x+ 18.75393 parsley ○ ○ R2 = 0.0169 0.3 ○ celery rose rugosa ○ poppy seed ○ 0.2 oolong ○ ○ oregano ○ ginger ○ ○ cumin perilla Enhancing effect on bitter taste(points) on bitter effect Enhancing 0.1 ○ ○ mace basil

○ ○ peppermint yuzu peel lemongrass 0 ○

-80 -60 -40 -20 0 20 40

GAD67 Activity (%) Fig. 5 Relationship for the effect of 17 food extracts on bitter taste sensation and that on GAD67 activity X axis indicates effects on GAD67 activity and Y axis indicates effect on the bitter taste. Correlation between the effects is shown as a line (R2=0.0169). 56 Association of GAD/ GABA and tastes

GABA are unlikely to participate in the involved in "contrast effect" known in umami taste transduction pathway, which cooking field. possibly is located in the type II cells.

Bitter Taste References Bitter taste is sensed by the type II cells since they express receptors for bitter taste. 1. Miura H (2006) Molecular We have examined if effects of food extracts mechanisms of taste reception and on bitter taste would be affected by GAD67 taste cell differentiation. Kagoshima activity and/or GABA. For this purpose, University Dental School Bulletin effects of each of the food extracts on 26:27-37. GAD67 relative activity ratio was plotted 2. Ninomiya Y (2007) Signal of taste against those on bitter taste potency (Fig. 5). and taste as modulators of food As a result, there seems to be no relationship information. Kagaku to Seibutsu (in between the GAD67 relative activity ratio Japanese) 45:419-425. and bitter taste potency (Fig. 5) for the 17 3. Royer SM & Kinnamon JC (1988) spice food extracts used for the sensory test. Ultrastructure of mouse foliate taste In addition, the degree of bitter taste was not buds: synaptic and nonsynaptic affected even when GABA was added (18, interactions between taste cells and 19). This suggests that bitter taste nerve fibers. J Comp Neurol transduction pathway may not be influenced 270(1):11-24, 58-19. by both GAD67 activity and GABA. 4. Takeda M (1976) An electron From the present study, it is suggested microscopic study on the that GAD67 and GABA may not take part in innervation in the taste buds of the sweet, umami or bitter taste signal mouse circumvallate papillae. Arch. transduction, where GAD 67 is projected in Histol. Jpn. 39:257-269. the type III cells and GABA is received by 5. Nakamura Y, Yanagawa Y, Obata K, the type II cells. Our present experiments & Ueno H (2006) GABA is have focused on the effects of the extracts produced in . Jpn J. Taste from spices, herbs and teas upon the defined Smell Res. 13:547-550. taste sensation. Since there was a limitation 6. Hisaki K, Hamano K, & Ueno H in the taste sensory testing method, we were (2012) About the role of GAD not able to clearly identify the involvement isoforms - Study from the effect on of GAD67 and GABA on the taste signal the taste and effect on GAD activity transduction; however, our present data does by food ingredients -. Jpn. J. Taste not deny any possibility that GABA may be Smell Res. 19:299-303. Sasaki, Hamano and Ueno 57

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