The TRPA1 Agonist, Methyl Syringate Suppresses Food Intake and Gastric Emptying
Citation: Kim MJ, Son HJ, Song SH, Jung M, Kim Y, et al. (
The TRPA1 Agonist, Methyl Syringate Suppresses Food Intake and Gastric Emptying
Min Jung Kim. 0
Hee Jin Son. 0
Seo Hyeon Song 0
Myungji Jung 0
Yiseul Kim 0
Mee-Ra Rhyu 0
Francois Blachier, National Institute of Agronomic Research, France
0 Division of Metabolism and Functionality Research, Korea Food Research Institute , Bundang-gu, Sungnam-si, Gyeonggi-do , South Korea
Transient receptor potential channel ankryn 1 (TRPA1) expressed in the gastrointestinal tract is associated with gastric motility, gastric emptying, and food intake. In this study, we investigated the effects of methyl syringate, a specific and selective TRPA1 agonist, on food intake, gastric emptying, and gut hormone levels in imprinting control region (ICR) mice. The administration of methyl syringate suppressed cumulative food intake and gastric emptying. In addition, treatment with ruthenium red (RR), a general cation channel blocker, and HC-030031, a selective TRPA1 antagonist, inhibited methyl syringate-induced reduction of food intake and delayed gastric emptying in ICR mice. Methyl syringate also increased plasma peptide YY (PYY) levels, but not glucagon-like peptide-1 (GLP-1) levels. The elevation in PYY was blocked by treatment with RR and HC-030031. The present findings indicate that methyl syringate regulates food intake and gastric emptying through a TRPA1-mediated pathway and, by extension, can contribute to weight suppression.
Funding: This study was supported by Korea Food Research Institute (E0131201). The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The gastrointestinal (GI) tract, the largest endocrine organ,
plays an important role in the regulation of energy homeostasis
and gastric emptying by coordinating appetite, food intake, and
body weight [1,2]. Gastric emptying is promoted by increased
food volume in the stomach, the presence of liquid food, and
protein. In contrast, the osmolarity of chime, fat, duodenal
distension, cold temperatures, and an acidic pH of 3.54 inhibit
gastric emptying [3,4]. Several GI hormones such as peptide YY
(PYY) and glucagon-like peptide 1 (GLP-1) also regulate gastric
emptying and food intake when secreted from L-cells in the ileum
into the circulation after food consumption . The effect of
GLP1 and PYY is late and long-term. GLP-1, found in the jejunum,
ileum, and colon, is stimulated by carbohydrate, protein, lipid, and
glucose concentration in blood . GLP-1 release
dose-dependently enhances insulin secretion from pancreas, insulin-sensitivity,
and insulin gene expression . GLP-1 also reduces glucagon
secretion from the pancreas, gastric secretion, gastric emptying,
and food intake. PYY increases electrolyte and water absorption in
the colon, while suppressing pancreatic secretion, gastric motility,
gastric emptying, and appetite [10,11].
Transient receptor potential cation channel, subfamily A,
member 1 (TRPA1), a member of the TRP family, is expressed
in sensory neurons and associated with somatosensation, such as
pain, cold, hot, and pungent irritants . The recent
identification of TRPA1 expression in other organs has revealed
additional functions. TRPA1 is highly expressed in
enteroendocrine cells in the GI tract of humans, mice, and rats, where it plays
an important role in delayed gastric emptying, secretion of gut
hormones and reduction of food intake [16,17]. TRPA1 responds
to various pungent ingredients in foods including allyl
isothiocyanate (AITC) in wasabi, allicin in garlic, benzyl isothiocyanate in
yellow mustard, isopropyl isothiocyanate in nasturtium seeds,
methyl isothiocyanate in capers, phenylethyl isothiocyanate in
Brussels sprouts, and ligustilide in celery (Apium graveolens L.) and
lovage (Levisticum officinale L) . Non-pungent compounds
such as capsiate and the fatty acids in royal jelly are TRPA1
We previously determined that methyl syringate is one of the
pungent ingredients in Kalopanax pictus Nakai (K. pictus) .
Methyl syringate increases cytosolic Ca2+ in human TRPA1
(hTRPA1)-transfected cells. This effect is inhibited by a general
cation channel blocker, ruthenium red (RR) or a selective TRPA1
blocker, HC-030031. Thus, methyl syringate is a specific and
selective activator of hTRPA1. Methyl syringate may be an
electrophile because it performs as an electon-transfer mediator in
Bratkovskaya et al. (2006) . The electrophilic TRPA1 agonists
such as AITC and lots of a,b-unsaturated aldehydes are covalently
modified cysteine residues in cytoplasmic domain of TRPA1 and
activates TRPA1 . Same as other electrophilic agonists,
methyl syringate may activate TRPA1 via a covalent pathway. In
this study, we investigated whether methyl syringate inhibits
gastric emptying by activating TRPA1 and stimulates the release
of gut hormones including PYY and GLP-1 in vivo in male
imprinting control region (ICR) mice.
Materials and Methods
Cinnamaldehyde, ruthenium red (RR), HC-030031, dimethyl
sulfoxide (DMSO), phenol red, methyl cellulose, and urethane
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Trichloroacetic acid was obtained from Junsei Chemical Co., Ltd.
(Chuo-ku, Tokyo) and methyl syringate from Alfa Aesar (MA,
USA). The structures of cinnamaldehyde and methyl syringate
were described in Figure 1.
Male ICR mice (3040 g) were obtained from Hanlim
Laboratory Animal Co. (Gyeonggi-do, South Korea). Mice were
housed in a room under a 12:12 h light: dark cycle (lights on 0700)
with ad libitum access to standard laboratory chow and tap water.
The room was maintained at 2262uC with relative humidity at
5961%. Prior to the experiments, all animals were housed in
individual cages and fasted overnight with access to water at all
times. The care and use of the animals followed our 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). All
surgery was performed under sodium pentobarbital anesthesia,
and all efforts were made to minimize suffering.
Evaluation of food consumption and gastric resection
Mice were divided into treatment groups (n = 4 per group) and
orally administered vehicle (1.5% methylcellulose) or 10 mg/kg
cinnamaldehyde or methyl syringate by gavage. Immediately after
treatment, the animals were given free access to their normal diet.
Food intake was recorded 28 h after treatment. To investigate the
effects of TRPA1 inhibition, 0.33 mg/kg RR or 0.15 mg/kg
HC030031 were administered with vehicle (1.5% methylcellulose) or
10 mg/kg cinnamaldehyde or methyl syringate. Food intake was
recorded at 2, 4, 6, 8, 24, and 28 h after treatment. After 28 h,
mice were sacrificed, the stomach of each mouse extracted, and
Figure 2. Effects of cinnamaldehyde (CALD) and methyl syringate (MS) on cumulative food intake. Vehicle (1.5% methyl cellulose),
CALD, or MS were administered orally to ICR mice at a dose of 10 mg/kg after fasting in the presence or absence of the TRPA1 antagonists RR and
HC-030031. Mice were allowed free access to normal food and water immediately after treatment. Changes in cumulative food intake were
monitored for 28 h. Data represent means 6 SEM (n = 4); *p,0.05 compared with the vehicle group by Dunnetts test.
Figure 4. Effects of cinnamaldehyde (CALD) and methyl syringate (MS) on gastric emptying. Phenol red was administered 5 min after
treatment with vehicle (1.5% methyl cellulose), CALD (0.180 mg/kg) and MS (0.110 mg/kg). Gastric emptying was evaluated by measuring the
quantity of phenol red retained in the stomach 15 min after administration. Columns and vertical bars represent means 6 SEM (n = 4); *p,0.05
compared with the vehicle control.
Evaluation of gastric emptying
Gastric emptying was performed 5 min after treatment with
individual drugs by administering a 0.05% phenol red solution
(0.15 ml/mouse). Fifteen minutes later, the mice were sacrificed by
intraperitoneal injection of 20% urethane. The stomach was
immediately removed and cut into several pieces in 2 ml of 0.1 N
NaOH, followed by addition of 0.02 ml 20% trichloroacetic acid.
The mixture was centrifuged for 10 min at 10,000 rpm, and the
supernatant (0.05 ml) added to 0.5 N NaOH (0.2 ml). The
absorbance of this mixed solution was measured with a
spectrometer at a wavelength of 560 nm. The gastric emptying
rate (%) was calculated = 100 (A/B)6100, where A is the amount
of phenol red remaining in the stomach 15 min after
administration of the phenol red solution, and B is the amount of phenol red
in the stomach immediately after administration of the phenol red
Fluorescence immunoassay of GLP-1 and PYY
Blood samples were collected in lavender Vacutainer tubes
containing EDTA at 0 and 15 min after treatment with vehicle,
cinnamaldehyde, or methyl syringate. Plasma isolated by
centrifugation was collected and stored at 220uC until analysis. Plasma
levels of GLP-1 and PYY were measured using a GLP-1
enzymelinked immunosorbent assay (ELISA) kit (Linco Research, St.
Charles, MO) or PYY ELISA kit (Phoenix Pharmaceuticals,
Mountain View, CA, USA). To investigate the effects of TRPA1
inhibition, 0.33 mg/kg RR or 0.15 mg/kg HC-030031 were
administered with vehicle (1.5% methylcellulose) or 10 mg/kg
cinnamaldehyde or methyl syringate and gut hormones measured
15 min after treatment. The relative changes in PYY and GLP-1
were calculated = A15 min/A0 min, where A15 min is the hormone
level in plasma 15 min after administration of the drugs, and A0
min is the hormone level in plasma at 0 min.
All results are expressed as means 6 standard error of the mean
(SEM). Data analysis was performed using the GraphPad Prism
software (GraphPad Software Inc., San Diego, CA, USA). The
results were analyzed by one-way analysis of variance (ANOVA)
and Dunnetts multiple range test.
Figure 5. Inhibitory effect of ruthenium red (RR) or HC-030031 on gastric emptying in mice treated with cinnamaldehyde (CALD) or
methyl syringate (MS). Phenol red was administered 5 min after treatment with vehicle (1.5% methyl cellulose), CALD, or MS at a dose of 10 mg/
kg in the presence or absence of RR (A) and HC-030031 (B). Gastric emptying was evaluated measuring the quantity of phenol red retained in the
stomach after 15 min. Columns and vertical bars represent the means 6 SEM (n = 4); *p,0.05 compared with the vehicle control.
Effects of cinnamaldehyde and methyl syringate on food
intake and remaining food in the stomach
The effects of cinnamaldehyde and methyl syringate on food
intake and food remaining in the stomach were investigated.
Cinnamaldehyde or methyl syringate was administered orally to
ICR mice at a dose of 10 mg/kg after fasting with the 1.5%
methyl cellulose vehicle used as a control. Subsequently, ICR mice
were allowed ad libitum access to food and water. Food intake was
measured at 2, 4, 6, 8, 24, and 28 h after treatment (Figure 2) and
the y-axis calculated as: D Cumulative Food Intake (g) = F2hFt,
where F2h is the amount of food remaining 2 h after
administration of methyl cellulose, cinnamaldehyde, or methyl syringate
solution and Ft is the amount of food remaining at each time point.
Food intake rapidly increased from 02 h with no significant
difference between the control and experimental group
(1.16360.038 g/h for 2 h, data not shown). In contrast, after
2 h, food intake decreased significantly. Therefore, cumulative
food intake for 28 h was evaluated 2 h after treatment.
Cumulative food intake in cinnamaldehyde-treated mice was
significantly reduced compared to the vehicle control 4 h after
treatment, while that in methyl syringate-administered mice was
significantly reduced 24 h after treatment. RR and HC-030031
efficiently blocked the effects of cinnamaldehyde at 28 h, but only
HC-030031 inhibited the effects of methyl syringate at 24 and
Figure 3 shows the amount of remaining food in the stomach
28 h after treatment. Normalized remaining foods in the stomach
were calculated as = Rsample/Rvehicle, where Rsample is the amount
of food remaining in the stomach 28 h after treatment with methyl
cellulose, cinnamaldehyde, or methyl syringate solution and
Rvehicle is the amount of food remaining in the stomach 28 h
after treatment with the methyl cellulose vehicle only. Normalized
remaining foods in the stomach of cinnamaldehyde- or methyl
syringate-treated mice were significantly decreased compared to
the control. Moreover, RR and HC-030031 reversed this effect in
that the amount of food remaining in the stomach of
cinnamaldehyde or methyl syringate-treated mice returned to control
Effect of cinnamaldehyde and methyl syringate on
The effects of cinnamaldehyde and methyl syringate on gastric
emptying were estimated in ICR mice. The body weights of ICR
mice were not significantly different between the control and
experimental groups (30.3260.24 g; p,0.05). The effects of
cinnamaldehyde (0.180 mg/kg) and methyl syringate (0.1
10 mg/kg) were compared with the 1.5% methyl cellulose vehicle
(Figure 4). Cinnamaldehyde dose-dependently delayed gastric
emptying, with a significant effect starting at 1 mg/kg and an IC50
value = 4.77 mg/kg. Methyl syringate also dose-dependently
delayed gastric emptying with ,70% reduction at a dose of
10 mg/kg compared to the control.
Inhibitory effect of RR or HC-030031 on delayed gastric
To investigate the effects of TRPA1 agonists mediated through
TRPA1 channels, we examined the effects of cinnamaldehyde on
mice treated with 0.33 mg/kg RR, a general cation channel
blocker or 0.15 mg/kg HC-030031, a selective TRPA1 antagonist.
RR and HC-030031 were administered to mice with 10 mg/kg
cinnamaldehyde or methyl syringate. In the presence of RR or
HC-030031, cinnamaldehyde or methyl syringate-induced
delayed gastric emptying returned to control levels (Figure 5). The
effects of RR and HC-030031 on cinnamaldehyde- or methyl
syringate-induced delayed gastric emptying were not significantly
different and neither RR nor HC-030031 alone affected basal
gastric emptying (data not shown).
Effects of cinnamaldehyde and methyl syringate on
plasma PYY and GLP-1 in mice
To investigate the involvement of cinnamaldehyde or methyl
syringate on regulation of food intake, plasma PYY and GLP-1
levels were analyzed (Figure 6). Cinnamaldehyde or methyl
syringate treatment increased plasma PYY levels 15 min after
treatment but did not affect GLP-1 levels. We confirmed that
cinnamaldehyde and methyl syringate elevated plasma PYY
through a TRPA1-mediated pathway by administering RR or
HC-030031. RR and HC-030031 blocked cinnamaldehyde- or
methyl-syringate-induced increases in plasma PYY levels and
reduced PYY in vehicle-treated animals.
Methyl syringate is a plant phenolic compound in Aspergillus
flavus, Aspergillus parasiticus, Betula alba, Kunzea ericoides (kanuka)
honeys, and Leptospermum scoparium (manuka) and has several
functions as a superoxide scavenger, an effective laccase mediator,
and an inhibitor of aflatoxin production . However, the
effects of methyl syringate on gastric functions remain unexplored.
In our previous report, we demonstrated that methyl syringate
from Kalopanax pictus activates TRPA1 and increases cytosolic Ca2+
concentrations in TRPA1-expressing cells .
TRPA1 expression has recently been identified in the stomach,
small intestine, and colon within the GI tract of the rat and mouse,
where it contributes to chemosensation, mechanosensation, GI
motility, delayed gastric emptying, and the regulation of food
intake [17,30] In rats, the TRPA1 agonists AITC, in the range of
0.0110 mg/kg, and cinnamaldehyde, in the range of 0.1
100 mg/kg, reduce gastric emptying. This effect is inhibited by
pretreatment with the cation blocker RR (1 mg/kg). Thus,
preventing TRPA1 controls gastric motility. Our results in ICR
mice correlate with these data in rats. We demonstrate that
cinnamaldehyde induced gastric emptying in a dose-dependent
manner, while RR and HC-030031 blocked the effect of
cinnamaldehyde on gastric emptying. Methyl syringate also
reduced gastric emptying in a dose-dependent manner, and RR
and HC-030031 suppressed delayed gastric emptying in ICR
The mechanism of TRPA1-mediated gastric emptying has not
been fully described. Doihara et al. (2009) suggested the
serotonergic pathway as one possible mechanism. In the GI tract, more
than 10 types of enteroendocrine cells exist, and TRPA1 is highly
expressed in enterochromaffin (EC) cells and endocrine cells.
Nutrients and non-nutrients stimulate EC cells to secrete serotonin
(5-HT), which controls GI motility and enhances secretory and
peristaltic reflexes, and endocrine cells to release cholecystokinin,
which contributes to delayed gastric emptying and reduces food
intake. The TRPA1 agonists, AITC and cinnamaldehyde, show
identical effects on 5-HT secreting cells and RIN14b cells . In
vitro, AITC and cinnamaldehyde covalently bind TRPA1 and
dose-dependently activate the TRPA1-mediated pathway to
increase Ca2+ influx. Subsequently, 5-HT is secreted from EC
cells and RIN14b cells. To confirm that TRPA1 mediates this
response, AITC or cinnamaldehyde-induced TRPA1 activation
was assessed using TRPA1-specific siRNA and RR. The
contribution of a serontonergic pathway was confirmed in male
Wistar rats using the tryptophan hydroxylase (TPH) inhibitor
pchlorophenylalanine (pCPA) and the 5-HT3 receptor antagonist
granisetron . TPH-1 contributes to 5-HT secretion in EC cells
via biosynthesis of 5-HT from tryptophan.
Other gastric hormones, including GLP-1 and PYY, influence
food consumption and energy intake. Nutrients, such as glucose
and fat, and non-nutrients, such as the neuromodulators
acetylcholine, GABA, and somatostatin, stimulate GLP-1 and
PYY secretion from endocrine L cells lining the small intestine into
the blood to affect energy intake. Two biologically active forms of
GLP-1, GLP-1 (737) and GLP-1 (736), exist with GLP-1 (736)
being the major circulating form in humans . Secreted GLP-1
binds to the GLP-1 receptor and stimulates downstream adenylyl
cyclase activation and cAMP production. Moreover, peripherally
injected GLP-1 induces increment c-fos expression in the brainstem
. GLP-1 also accelerates glucose-stimulated insulin secretion
and suppresses glucagon secretion, gastric emptying, and food
consumption. PYY has two circulating forms, PYY (136) and
PYY (336) [33,34]. The N-terminal tyrosine-proline residues
from PYY (136) are cleaved by the enzyme dipeptidyl peptidase-4
(DPP-4) to produce the major circulating form, PYY (336). Many
studies suggest that PYY (336) influences feeding via the
hypothalamus [35,36]. PYY (336) binding to the hypothalamic
Y2 receptor activates c-fos, a marker of neuronal activation, and
reduces food intake. This anorexic action has been demonstrated
in mice intraperitoneally administered PYY (336) and in humans
. Additionally, increased GLP-1 and PYY in plasma delay
We found that cinnamaldehyde and methyl syringate elevated
plasma PYY levels in male ICR mice fed a liquid meal of 1.5%
methylcellulose. Plasma PYY levels were increased with 0.1
1 mg/kg cinnamaldehyde, but decreased in the range from 1.0
80 mg/kg cinnamaldehyde. In contrast, cinnamaldehyde and
methyl syringate had no significant effects on plasma GLP-1 levels.
Theoretically, cinnamaldehyde and methyl syringate identically
elevate or inhibit these hormones since PYY and GLP-1 are
usually co-secreted from L cells. However, only plasma PYY was
increased, which could be explained by the plasma half-life of PYY
and GLP-1. Plasma PYY is elevated after 15 min, while plasma
GLP-1 level is increased 10 min after food intake [38,39].
However, the timing and magnitude of PYY and GLP-1 secretion
depend on the composition and content of the meal . For
example, whey protein, which is absorbed faster than casein,
elicited a greater GLP-1 response than casein . Methyl
cellulose, used in this study as a control, is a synthetic fiber derived
from cellulose and had no effect on appetite. The interaction rate
between cinnamaldehyde or methyl syringate in liquid form and
TRPA1 in the GI tract is unknown. Also, GLP-1 showed a shorter
half-life (,2 min) than PYY (.8 min). Therefore, GLP-1 may not
be detected in collected plasma.
In summary, methyl syringate, a TRPA1 activator, significantly
suppressed food intake and delayed gastric emptying by elevating
plasma PYY in male ICR mice. This can reduce the time
necessary to perceive gastric fullness and so result in weight loss.
Obesity is closely connected to type 2 diabetes, heart disease,
several types of cancer, and osteoarthritis. Delayed gastric
emptying is one method of reducing body weight. The current
study suggests that methyl syringate, which reduced food intake,
gastric emptying, and secretion of gut hormones, may contribute
to weight suppression.
Conceived and designed the experiments: HJS MJK MRR. Performed the
experiments: HJS SHS MJ MJK YK. Analyzed the data: MJK HJS SHS
MRR. Contributed reagents/materials/analysis tools: HJS SHS MJ YK
MJK MRR. Wrote the paper: MJK MRR.
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