Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2
Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2
Jaewon Shim 0 1 2
Hee Jin Son 0 1 2
Yiseul Kim 0 1 2
Ki Hwa Kim 0 1 2
Jung Tae Kim 0 1 2
Hana Moon 0 1 2
Min Jung Kim 0 1 2
Takumi Misaka 0 1 2
Mee-Ra Rhyu 0 1 2
0 1 Division of Creative Food Science for Health, Korea Food Research Institute , Bundang-gu, Sungnam-si, Gyeonggi-do , Republic of Korea, 2 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo , Bunkyo-ku, Tokyo , Japan
1 Funding: This study was supported by Korea Food Research Institute (E0131201, E0143043494). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
2 Academic Editor: Keiko Abe, The University of Tokyo , JAPAN
Although the five basic taste qualities-sweet, sour, bitter, salty and umami-can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5'-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/ hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.
Competing Interests: The authors have declared
that no competing interests exist.
Most food products comprise of multiple mixtures of tastants. Animals integrate and unify the
information regarding each separate taste and decide on their feeding behavior. Much research
has focused on and described the interactions between taste modalities . However, these
studies are restricted to observations of phenotype and phenomenon and the detailed
molecular and cellular mechanisms have not been fully investigated.
These interactions occur not only at the level of neuronal transduction but also at level of
taste receptor [5, 6]. This crosstalk probably results from multiple mode of ligand binding to
taste receptors. For example, a recent study revealed that binding of amiloride, a type of salt
sensing reducer, to sweet receptors inhibited their responses .
Taste-taste interactions among the basic tastes have been investigated [1, 2]. Umami also
interacts with the other tastes. Kemp and Beauchamp  concluded that at moderate/high
concentrations of monosodium glutamate (MSG), sweet and bitter tastes were suppressed.
Conversely, Woskow  reported that 5-ribonucleotides which exhibit umami taste enhanced
sweetness and saltiness at moderate concentrations, while sourness and bitterness were
suppressed. Since these observations are based on behavioral indices, it remains to be elucidated
whether the increase or decrease of sweetness caused by umami compounds occur at sweet
taste receptor cells.
Sweet taste receptors in mammals are heterodimeric receptor complexes that comprise of
T1R2 (taste type 1 receptor 2) and T1R3 (taste type 1 receptor 3) . These receptors have
a transmembrane domain (TMD) and a large extracellular domain (ECD), which is composed
of a large extracellular venus flytrap domain (VFD) and a short cysteine-rich domain (CRD)
[12,13]. Several reports show that the ECD is responsible for agonist recognition .
Aspartame and acesulfame K are recognized by the ECD of human T1R2 (hT1R2). In contrast,
TMD of human T1R3 (hT1R3) is responsible for the recognition of cyclamate and for binding
of lactisole which acts as a noncompetitive inhibitor .
In this study, we investigated the relationship between umami compoundssuch as MSG
and glutamyl dipeptidesand sweet receptors at the receptor level. We showed that umami
compounds inhibited the response of sweet receptors in a manner dependent on the sweet
receptor agonist. In addition, we provide the evidence that umami compound might inhibit
agonist binding at T1R2 in allosteric manner.
Sucrose, acesulfame K, aspartame, cyclamate and MSG (L-glutamic acid monosodium salt
hydrate) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glu-Glu, Glu-Asp were
synthesized from Lugen Sci (Seoul, Republic of Korea). Cell culture media were obtained from Life
Technologies, Inc. (Grand Island, NY, USA).
Cell culture and transfection
Flp-In 293 cells stably expressing hT1R2, hT1R3 and Gustducin (wild-type) and hT1R2,
hT1R3(F778A) and Gustducin (mutant) were prepared as described previously [7,21]. The
hT1R2/hT1R3-expressing cells were maintained in Dulbeccos modified Eagles medium
(DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen)
and 0.2% hygromycin B (Invitrogen). All cells were incubated at 37C in a humidified
atmosphere containing 5% CO2. Cultured hT1R2/hT1R3-expressing cells were seeded onto 96-well
black-wall plates for 24 h prior to their use in experiments.
Ca2+ imaging of the responses of hT1R2 and hT1R3-expressing cells
hT1R2/hT1R3 stably expressing cells were seeded onto 96-well black-wall imaging plates (BD
Falcon Labware, Franklin Lakes, NJ, USA) for 24 h prior to their use in experiments. After
24 h, the cells were washed with assay buffer (130 mM NaCl, 10 mM glucose, 5 mM KCl, 2
mM CaCl2, 1.2 mM MgCl2 and 10 mM HEPES; pH 7.4) and loaded with the Ca2+ indicator
dye Fluo-4 (5 M; Invitrogen) in assay buffer for 30 min at 27C. The cells were rinsed with
assay buffer, incubated in 100 L of assay buffer for > 10 min and then treated with ligand by
adding 100 L of the ligand solution. Fluo-4 was excited with the 486nm, and fluorescence was
measured at wavelengths >515nm. [Ca2+]i was read into a computer-controlled filter changer
(Lambda DG4; Sutter Instrument Co., San Rafael, CA, USA), an Andor Luca CCD camera
(Andor Technology, Belfast, Northern Ireland) and an inverted fluorescence microscope
(IX71; Olympus, Tokyo, Japan). Images were recorded at 3-s intervals and were analyzed using the
MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).
Measurement of cytosolic Ca2+ levels in hT1R2/hT1R3-expressing cells
using a fluorescence plate reader
hT1R2/hT1R3 stably expressing cells were seeded onto 96-well black-wall CellBIND Surface
plates (Corning Inc., Corning, NY, USA) for 24 h before the assay. The cells were loaded with
5 M Calcium-4 (Molecular Probes, Eugene, OR, USA) in assay buffer for 45 min at 27C.
Subsequently, the cytosolic Ca2+ concentration was measured using a Flex Station III microplate
reader (Molecular Devices). Sample solutions were loaded after a 17-s baseline scan and
ligand-induced changes in fluorescence intensity (excitation, 485 nm; emission, 525 nm; cutoff,
515 nm) were monitored at 2.1-s intervals for 120 s. The response of each well is presented as
the change in relative fluorescence units (RFU), which was defined as the maximum minus
the minimum fluorescence value. All experiments were performed at least three times.
To examine interaction between sweet and umami taste signaling at the receptor level, we
evaluated the response of sweet receptor cells after treatment with various combinations of umami
compounds and sucrose. The response of sweet receptor cells was monitored using the Flex
system with the Ca4 dye. When MSG was co-applied with sucrose, the induced response of
sweet receptor cells by sucrose was attenuated (Fig 1A). Other umami compounds; i.e.,
glutamate-glutamate (Glu-Glu) dipeptide, and glutamate-aspartate (Glu-Asp) dipeptide, also
reduced significantly the response of sweet receptor cells (Fig 1B and 1C). To investigate whether
all dipeptides might affect sweet receptor activity, we tested the sweet response with
glycineglycine (Gly-Gly) dipeptide which is well known as the taste-less dipeptides. Gly-Gly did not
show inhibitory effect as induction of sweet receptor by sucrose (Fig 1E). Attenuated effect by
MSG or umami dipeptides got clear as strong induction by treatment of high concentration of
sucrose. Since treatment of sucrose in higher concentration than 150 mM had the effect on
only Flp-In 293 cells without expressing sweet receptors, we did not perform the experiments
with higher than 150 mM sucrose. The inhibitory effect of MSG and dipeptides showed
dosedependency (Fig 1A1C). Because high osmolarity with MSG might cause inhibitory effect of
sucrose response, we performed the control experiment using high concentration of Mannitol
to mimic high osmolarity . 50 mM of Mannitol causes the inhibited sucrose response.
However, it seems to be ignorable because the inhibitory rate by Mannitol is much less than
Fig 1. Inhibition of sucrose-induced calcium responses in hT1R2/hT1R3-expressing cells by MSG (A), Glu-Glu (B), Glu-Asp (C), Gly-Gly (E), IMP
(F), and GMP (G). Glu-Glu and Glu-Asp are umami peptides and Gly-Gly is tasteless peptide. Inhibitory effect of umami compounds evaluated using the Flex
system. Co-application of MSG or glutamyl dipeptides with sucrose inhibited the response of sweet receptor cells to sucrose. However, co-application of the
5-ribonucleotides IMP and GMP with sucrose did not inhibit the response of sweet receptor cells to sucrose. (D) Osmolarity effects in hT1R2/
hT1R3-expressing cells. (H) Sucrose response with dipeptides adjusted to pH 7.4. The value of y-axis means the ratio which the value with agonists and/or
compounds normalized by the value without agonist. Asterisk *, **, *** stands for p<0.05, p<0.01, p<0.001, respectively. (I) Ca2+ cell images using fluo-4
dye is captured at 30 seconds later after co-applied sucrose and/or umami-compounds. Responded cell emitted the green fluorescence signals. Similar to
the results of the Flex system, the response of sweet receptor cells to sucrose was attenuated by the co-application of Glu-Glu, Glu-Asp or MSG. (J) The ratio
of responding cells to total cells.
that by MSG (Fig 1D). In addition, low pH with acidic dipeptides might cause inhibited sucrose
response. To remove the pH effect, we checked the response of sucrose with dipeptides revised
to pH 7.4 using NaOH . They are still highly effective in the inhibited sucrose response as
much as unrevised dipeptides are (Fig 1H). Sweet receptor signaling finally increases cellular
Ca2+ level via cAMP or IP3 production . Although the change of cellular Ca2+ level was
detected by Flex system, this phenomenon was more convinced by visualization of cellular Ca2+
level using imaging system with the Fluo-4 dye (Fig 1I and 1J). Visualized images using Fluo-4
dye also showed the inhibited sucrose response by MSG or dipeptides. These results were
quantified by measuring the ratio of fluorescent cells by total cells in the Fig 1J. To investigate
whether all umami compounds could inhibit sweet receptor activity, we tested the response of
sweet receptor cells after treatment with 5-ribonucleotide umami agonists such as IMP and
GMP (Fig 1F and 1G). However, the 5-ribonucleotide umami compounds did not reduce the
Fig 2. The inhibitory effect by umami compounds was dependent on the sweet receptor agonist. The responses of hT1R2/hT1R3-expressing cells
were measured after co-application of umami compounds (30 mM MSG and 1 mM Glu-Glu) with other agonist; acesulfame K (A), aspartame (B) or
cyclamate (C). Inhibitory effect of sweet receptor by MSG or umami dipeptide (Glu-Glu) was not shown at induction by aspartame or cyclamate. Asterisk *,
**, *** stands for p<0.05, p<0.01, p<0.001, respectively.
response of sweet receptor cells to sucrose. Since 5-ribonucleotide affects the T1R1 receptor, a
component of the umami receptor complex, understandably the agonists had no effect on the
T1R2/T1R3 sweet receptor complex.
To investigate whether MSG and umami-dipeptide inhibit agonistreceptor binding, we
tested three other sweet receptor agonists: acesulfame K, aspartame and cyclamate. Acesulfame K
and aspartame activate the sweet receptor in the ATD of T1R2, whereas cyclamate affects the
TMD of T1R3. Induction of sweet receptor cells by acesulfame K was inhibited by Glu-Glu and
MSG, similar to sucrose (Fig 2A). Compared with these agonists, treatment of Glu-Glu, MSG
did not inhibit the cyclamate- or aspartame-induced response of sweet receptor cells (Fig 2B
and 2C). Although aspartame affects the ATD of T1R2, a recent study suggested that aspartame
uses different binding sites than acesulfame K . These results suggest that MSG and umami
dipeptides affect specific regions of the ATD of T1R2 to inhibit agonist binding.
Lactisole is the well-known antagonist by affecting the TMD of T1R3 . To evaluate whether
umami compounds works on the TMD of T1R3 for inhibition of sweet receptors, we
performed a combination treatment of Glu-Glu and/or lactisole with sucrose. If umami
compounds and lactisole share a common interacting surface in the TMD of T1R3, their inhibitory
effect might be competitive. If umami compounds works on another site, the inhibitor effects
would be additive or synergistic. Since no inhibitory effect of Glu-Glu on cyclamate (which
also affects T1R3 TMD) was observed (Fig 2C), we hypothesized that the inhibitory
mechanism of Glu-Glu might be different from that of lactisole. As expected, combination treatment
of Glu-Glu and lactisole showed a reduced response to sucrose compared to that of each
compound alone (Fig 3A). Additionally, we evaluated the inhibitory effect of the response of sweet
receptor cells containing a F778A point mutation in T1R3. As a previous report , sweet
Fig 3. The target site of the umami peptide in sweet receptors was distinct from that of lactisole (A) The synergistically reduced response of sweet
receptor cells upon application of sucrose. Co-application of 1 mM Glu-Glu with indicated concentration of lactisole (Lac) synergistically inhibited the sweet
response. Asterisk *, **, *** stands for p<0.05, p<0.01, p<0.001, respectively. (B) The response of F778A mutant to cyclamate and sucrose. F778A means
a point mutation at residue 778 of hT1R3 resulting in substitution of phenylalanine for alanine. (C) The response to the co-application of sucrose and Glu-Glu
in cells expressing F778A mutant human sweet-taste receptors. (D) Imaging of co-application of 100 mM sucrose with 1 mM Glu-Glu or 50 mM MSG in cells
with the F778A mutant receptor. (E) The ratio of responding cells to total cells.
receptor cells containing this point mutation showed defective cyclamate induction, whereas it
did not show defective sucrose induction (Fig 3B). Combination treatment with Glu-Glu and
sucrose in cells with this mutant receptor still showed the inhibitory effect of Glu-Glu (Fig 3C).
These results indicate that the effect of Glu-Glu is not mediated via F778 on T1R3 TMD. Even
though the results from Flex system were consistent and convincing, confirmation by the other
system such as imaging system using the Fluo-4 dye would make our results stronger. This
phenomenon was confirmed using an imaging system that could detect Ca2+ influx caused by
receptor activity (Fig 3D). These results were quantified by measuring the ratio of fluorescent
cells by total cells (Fig 3E).
Although much research has focused on taste-taste interactions, few studies have investigated
the combination of sweet and umami tastants. Sako et al.  measured the response of the
chorda tympani (CT) nerve of rodents to sucrose and MSG either alone or in combination
with MSG, and reported that the combination of the sweeteners and MSG exerted synergistic
effects. The enhancement was suggested to be due to colocalization of sweet and umami
receptors in the same taste receptor cells (TRCs). Kemp and Beauchamp  reported changes
in other tastes with the application of MSG based on behavioral testing. They showed that at
moderate/high concentrations of MSG, sweet and bitter were suppressed; moreover, at a high
concentration of MSG, the saltiness of NaCl was enhanced. However, these studies were based
on the observation of behaviors and do not provide information regarding the underlying
In this study, we investigated the relationship between umami compounds and the sweet
receptor T1R2 and T1R3 complexes. Interestingly, MSG and glutamyl peptides inhibited the
responses of sweet receptors. The reduced response of sweet receptor cells caused by umami
compounds was dependent on the agonist type. Activation of sweet receptors by sucrose or
acesulfame K was attenuated by umami compounds, whereas no such inhibition was detected
when activated by cyclamate or aspartame. Ligands that are inhibited by umami compounds
have a common activation site: the ECD of T1R2. Activation of sweet receptors by aspartame
was not blocked, although aspartame induces the sweet receptor complex via the ECD of
T1R2. This result can be explained based on the findings of a recent study . Masuda et al.
analyzed the binding modes between human sweet receptors and sweet compounds and
defined the T1R2 residues used in agonist binding. In that report, sweet compounds were
grouped according to the hT1R2 residues required for their recognition. Acesulfame K requires
the R383, D142 and E382 residues of hT1R2 to activate sweet taste receptors, whereas
Aspartame recognition requires the E302 and S144 residues of hT1R2. Because activation of sweet
receptor with Acesulfame K was inhibited by umami compounds, but not with Aspartame, this
allosteric hindrance of umami compounds might affect the R383 and D142 residues of the
ECD of hT1R2.
Both of hT1R1 and hT1R2 belong to class C G protein-coupled receptors, thus sharing
conserved structural features . Each of these receptors possesses a large extracellular Venus
flytrap domain (VFT) that is linked to a small extracellular cysteine-rich domain (CRD) and a
seven-transmembrane domain (TMD). VFT consists of two lobes and the ligand binding site is
located in a hinge region between the two lobes . hT1R1 shows amino acid sequence
homology with hT1R2 . Based on well-known fact that MSG interact with VFT of hT1R1,
this structural similarity supply the possibility that MSG might influence on hT1R2.
An alternative mechanistic hypothesis is that chemical interactions occur in the mixture
before it comes into contact with the taste receptors. However, this is unlikely because the two
structurally different types of agonist tested showed a common inhibitory effect.
In our study the underlying molecular mechanism could not be determined. Since we used
only in vitro systems, examination of the interaction between umami compounds and sweet
receptors might require different conditions. Additionally, the T1R2 residues to which umami
peptides bind should be explored in further studies. Despite the limitations, our study is
important because it is the first report to describe the relationship between sweet receptors and
umami compounds at the molecular and receptor levels.
Conceived and designed the experiments: JS MRR. Performed the experiments: JS HJS YK
KHK JTK HM MJK. Analyzed the data: JS HJS YK MJK MRR. Contributed reagents/materials/
analysis tools: HJS YK KHK TM. Wrote the paper: JS MRR.
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