allyl isothiocyanate (AITC), a natural compound in plants belonging to the family Cruciferae, is known as the pungent ingredient in mustard, horseradish, and wasabi. Ingestion of spicy or pungent compounds in foods has traditionally been thought to enhance thermogenesis and energy expenditure. These effects of capsaicin, one of the spicy compounds in foods, have been well studied; however, it is unclear whether the similar pungent compound AITC affects thermogenesis and energy metabolism.

The transient receptor potential (TRP) channel family is composed of a wide variety of cation-permeable channels and shows a great diversity of activation mechanisms. TRPV1 and TRPA1 are cation channels belonging to the TRP channel family that are activated by high (>43°C) (8, 55) and low (<18°C) (4, 52) nociceptive temperatures, respectively; therefore, they are termed thermosensitive TRP channels. Interestingly, they are also activated by spicy or pungent compounds in foods. For example, TRPV1 is activated by ingredients of spicy foods such as hot pepper (capsaicin) (8), pepper (piperine) (38), and ginger (gingerols and shogaols) (10, 46). TRPA1 is activated by ingredients of pungent foods such as cinnamon (cinnamaldehyde) (4), and garlic (allicin) (35). AITC has also been reported to activate TRPA1 (24).

Recent studies have indicated that activation of TRPV1 or TRPA1 is involved not only in nociception and thermosensation but also in thermoregulation and energy metabolism (3, 22, 28, 36, 44, 51). We previously demonstrated that intragastric administration of TRPV1 agonists (capsaicin and capsiate) enhances oxygen consumption (V̇o₂) and increases core body temperature (43–45). Furthermore, studies of TRPV1 knockout (KO) mice and TRPV1 antagonists revealed that such effects were mediated by TRPV1 (28, 43). Intragastric administration of TRPA1 agonists (AITC and cinnamaldehyde) enhances thermogenesis in interscapular brown adipose tissue (36, 56). Intravenous administration of both TRPV1 and TRPA1 agonists also enhances adrenalin secretion from the adrenal medulla through the central nervous system (23, 30, 32, 58, 59), and it enhances metabolisms in skeletal muscles (31, 37). From these facts, it is expected that activation of TRPV1 or TRPA1 might affect thermogenesis and energy metabolism.

The relationship between TRP channels and energy metabolism has been well studied; however, there have been few reports on energy substrate utilization, i.e., whether carbohydrate or fat is preferentially metabolized by TRP channel activation. Recent studies suggest that TRPV1 dysfunction is associated with the development of diabetes and obesity (54), and several studies indicate a relationship between TRPV1 and blood glucose metabolism (1, 15–17). If we could reveal the relationship between TRP channel activation and the regulation of substrate utilization, we could control energy metabolism depending on the type of metabolic disorder by using TRP channel agonists. This kind of information is expected to be very useful for our modern lifestyle, with excessive energy intake and reduced physical activity.

In the present study, we investigated the effect of intragastric administration of AITC, which is generally regarded as a TRPA1 agonist, on changes in substrate oxidation. We also investigated the relationship between TRP channels and substrate utilization by AITC by using TRPA1 KO and TRPV1 KO mice. Together with examination of TRPV1-overexpressing cells, we examined how AITC enhanced substrate utilization.

MATERIALS AND METHODS

Animals.

Male C57BL/6 mice (6–9 wk old; Japan SLC, Shizuoka, Japan) were used. Mutant TRPV1-null mice and TRPA1-null mice were generously provided by Dr. D. Julius (University of California, San Francisco, CA). The procedure of mutant mice was reported in previous literature (5, 7). Mutant mice were back-crossed into the C57BL/6 genetic background. The mice were housed in a vivarium maintained at 23 ± 2°C under a 12:12-h light-dark cycle (lights on 0600–1800 h) with free access to a commercial standard laboratory chow (MF; Oriental Yeast, Tokyo, Japan) and drinking water. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Kyoto University and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Respiratory gas analysis.

The mice were kept individually in a chamber for 3 h to attain a constant respiratory exchange ratio (RER). A sample was intragastrically administered, and the expired air was analyzed. The oxidation of total fatty acids and carbohydrates was computed on the basis of (V̇o₂) and carbon dioxide production (V̇co₂). Gas analysis was performed using an open circuit metabolic gas analysis system connected directly to a mass spectrometer (model Arco2000; ArcoSystem, Chiba, Japan). The gas analysis system has been described in detail elsewhere (20, 21). Briefly, each metabolic chamber had a 72-cm² floor and was 6 cm in height. Room air was pumped through the chambers at a rate of 0.5 l/min. Expired air was dried in a cotton-thin column and then directed to an O₂/CO₂ analyzer for mass spectrometry.

On the basis of the volume of CO₂ production per unit of time (l/min) V̇co₂ and V̇o₂, total glucose, and lipid oxidation were calculated using the stoichiometric equations of Frayn (13) as follows: total fatty acid oxidation = 1.67 V̇co₂ − 1.67 V̇o₂ and carbohydrate oxidation = 4.55 V̇o₂ − 3.21 V̇co₂.

Materials.

DMEM and other cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Fura-2 acetoxymethyl ester (Fura-2 AM) was obtained from Dojindo Chemicals (Kumamoto, Japan). Doxycycline was obtained from BD Clontech (Mountain View, CA). Capsaicin and capsazepine were obtained from Sigma (St. Louis, MO). AITC was purchased from Nacalai Tesque (Kyoto, Japan). Cinnamaldehyde was obtained from Wako Chemicals (Tokyo, Japan). For intragastric administration, both AITC and cinnamaldehyde were diluted in saline containing 3% ethanol and 10% Tween 80.

Cell culture.

The cDNA encoding rat TRPV1 was kindly provided from D. Julius. The entire coding regions of rat TRPV1 were subcloned into pcDNA5/FRT to yield pTRPV1FRT. To establish doxycycline-inducible TRPV1-expressing cells, Flp-In-T-Rex-293 cells (Invitrogen) were cotransfected with pTRPV1FRT and pOG44 (BD Clontech) by using lipofectamine 2000 (Invitrogen). At 24 h after transfection, transfected cells were selected for hygramycin B (400 μg/ml) resistance by using medium containing. After 14 days, hygromycin-resistant clones were collected and combined. TRPV1 expression was induced by incubation with doxycycline (1 μg/ml) for 12–24 h before experiments.

Cells were routinely maintained in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO₂. They were passaged twice a week at a ratio of 1:5 to maintain an exponentially growing state. Experiments were performed 2 days after each passage.

Intracellular calcium ion concentration measurement.

Intracellular calcium ion concentration ([Ca²⁺]_i) was measured with a Fura-2 AM imaging method. Cells on dishes were detached using Ca²⁺-free PBS containing 0.5 mM EDTA and then collected by centrifugation. The collected cells were washed with PBS and resuspended in Krebs solution (containing in mM: 135 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, adjusted to pH 7.4 with NaOH) containing the cytoplasmic calcium indicator Fura-2 AM (2 μM) at 37°C for 45 min. After washing with nominal Ca²⁺-free Krebs solution containing 1 mM EGTA, the cells were stocked in Krebs solution or nominal Ca²⁺-free Krebs solution at 25°C for 5 min. The cells were suspended at a concentration of 3 × 10⁵ cells/ml in Krebs solution.

A cuvette containing Fura-2-loaded cells was placed in a fluorospectrophotometer (model CAF-110; Jasco, Tokyo, Japan). After incubation with stirring at 37°C for at least 1 min, the test compound was added. Time-dependent changes in fluorescence (excitation wavelength set at 340 of 380 nm and emission wavelength at 550 nm) were recorded and analyzed using PowerLab system MacLab/4e and Chart 4 (AD Instruments). The fluorescence ratio (340/380) was converted according to the equation published by Grynkiewicz et al. (18), where R_max and R_min were determined using 0.4% Triton X-100 and 20 mM EDTA, respectively. The effective dissociation constant for Fura-2 at 37°C was 224 nM.

TRPV1 antagonist capsazepine was added with AITC. When inhibition by capsazepine is measured, 10 μM capsazepine was added to 10 and 100 μM AITC and 0.1 and 1 μM capsaicin. The test compounds were prepared in DMSO and add to the loading solution (final DMSO concentration, 0.2%).

[Ca²⁺]_i measurement of dorsal root ganglia neuron.

TRPA1-null mice (7–9 wk old) and wild-type (WT) mice (C57/BL6) were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg) and killed by decapitation. The dorsal root ganglia (DRG) were removed and were minced in HBSS. The cells were dissociated with collagenase (5.0 mg/ml) and dispase (5.0 mg/ml) for 1 h and separated on a percoll gradient (12). Cells were plated onto collagen-I-coated coverslips in DMEM containing 10% FBS and supplemented with penicillin and streptomycin (Gibco) for 2 h.

Cells on the collagen-I-coated coverslips were loaded with Fura-2 AM (2 μM) at 37°C for 1 h. After being washed with Krebs solution, the coverslip was put on a recording chamber (Warner Instruments, Hamden, CT) mounted on the stage of an upright fluorescence microscope (model BS50WI; Olympus). Cells in the chamber were perfused with Krebs solution. AITC was diluted to the indicated concentration just before the experiment. Ca²⁺ imaging was performed by using Fura-2 with a digital image analysis system (AQUACOSMOS; Hamamatsu Photonics, Hamamatsu). The Fura-2 fluorescence emission, which was caused by excitation at 340 and 380 nm, was measured at 510 nm (F340 and F380). The images of the cells were captured every 4 s. The signal ratio at F340:F380 was converted into [Ca²⁺]_i levels using an in vivo calibration with 1 μm ionomycin (60).

Vagotomy procedure.

Mice were anesthetized using pentobarbital sodium (50 mg/kg), and following an upper midline laparotomy the stomach and lower esophagus were visualized. The skin and abdominal wall were incised along the ventral midline, and the intestines were moved aside to allow access to the left lateral lobe of the liver and the stomach. The left lateral lobe of the liver was gently retracted, and a ligature was placed around the esophagus at its entrance to the stomach, which allowed access to the esophagus. The stomach was gently pulled down beneath the diaphragm to clearly expose both vagal trunks, which were then transected. All neural and connective tissue surrounding the esophagus were removed to ensure transection of all small vagal branches.

To confirm completeness of vagotomy, a food intake analysis test was performed based on the satiety effect of cholecystokinin-octapeptide (CCK-8; Sigma) (26, 41). Animals were deprived of food for 8 h and then received an intraperitoneal injection of 8 μg/kg CCK-8 per mouse. Food intake over a 30-min period was measured. Subdiaphragmatic vagotomy abolished the satiety effect of CCK-8 so the food intake in vagotomized mice was similar to saline-injected animals (vagotomy/saline 0.375 ± 0.035 g vs. vagotomy/CCK 0.318 ± 0.035 g food intake). Any vagotomized animal that decreased their food intake significantly was excluded from the study.

Data analysis.

All values are presented as means ± SE. The effect of intragastric administration of AITC or cinnamaldehyde on RER, V̇o₂, carbohydrate oxidation, and fat oxidation were examined by two-way repeated-measures ANOVA with Bonferroni post hoc test (Prism 4.0; GraphPad Software, San Diego, CA). In TRPA1 or TRPV1 KO mice, the effects of intragastric administration of AITC on RER, V̇o₂, carbohydrate oxidation, and fat oxidation were examined by two-way repeated-measures ANOVA followed by unpaired t-test. The effects of intragastric administration of AITC on average RER and cumulative V̇o₂, carbohydrate oxidation, and fat oxidation for 2 h were examined by one-way ANOVA (see Fig. 2). Tukey's test was used as a post hoc test. In TRPA1 or TRPV1 KO mice, the effects of intragastric administration of AITC on average RER and cumulative V̇o₂, carbohydrate oxidation, and fat oxidation for 2 h (see Figs. 4 and 9) were examined by using an unpaired t-test. The effect of intraperitoneal administration of AITC to mice or intragastric administration of AITC to vagotomized mice on RER and V̇o₂, were examined by two-way repeated-measures ANOVA followed by unpaired t-test.

RESULTS

Effects of intragastric administration of AITC on changes in energy substrate utilization.

We measured RER after intragastric administration of AITC by indirect calorimetry. Mice were fasted for 3 h before the experiment to avoid the effect of components in diet (MF; Oriental Yeast) on its digestion and absorption on its metabolism. Intragastric administration of AITC dose dependently elevated RER for 2 h after administration compared with vehicle (Fig. 1). Significant differences were observed at a dose of 25 mg/kg but not at a dose of 5 mg/kg (data not shown). At 25 mg/kg dose, the peak RER elevation was observed at 30 min after administration; at 50 mg/kg dose, it was observed at 60 min after administration. AITC dose dependently increased carbohydrate oxidation for 2 h after administration compared with vehicle. In contrast, AITC dose dependently decreased fat oxidation for 2 h after administration. V̇o₂ was slightly higher for 20–40 min after administration in the AITC-treated group than in the vehicle-treated group. However, there was no significant difference between each group in the cumulative total V̇o₂ for 2 h after administration (Fig. 2). Since a dose of 25 mg/kg AITC was regarded as a sufficient dose to affect metabolism, it was used in further experiments.

Contribution of TRPA1 to changes in energy substrate utilization.

AITC is generally regarded as a TRPA1 agonist. To examine the contribution of TRPA1 to changes in energy substrate utilization induced by AITC administration, we administered AITC to TRPA1 KO mice. In TRPA1 KO mice, intragastric administration of AITC significantly elevated RER for 40–90 min after administration compared with vehicle administration (Fig. 3). This finding was similar to that observed in WT mice. AITC increased carbohydrate oxidation for 40–90 min after administration comparing with vehicle. AITC also decreased fat oxidation, although two-way repeated-measures ANOVA showed no significant difference. However, there was a significant difference between each group in the cumulative total fat oxidation for 2 h after administration (Fig. 4). V̇o₂ was not affected for at least 2 h after administration.

Effects of intragastric administration of cinnamaldehyde on changes in energy substrate utilization.

We used another TRPA1 agonist, cinnamaldehyde, to examine the contribution of TRPA1 to changes in energy substrate utilization. Intragastric administration of cinnamaldehyde did not affect RER, V̇o₂, carbohydrate oxidation, and fat oxidation for at least 2 h after administration at a dose below 100 mg/kg (Fig. 5).

Activation of TRPV1 by AITC.

It has been reported that TRPA1 is coexpressed with TRPV1 in a subset of small-to-medium diameter peripheral sensory neurons (24, 52), and that allicin, structurally similar to AITC, activates TRPV1 (48). Consequently, we considered that TRPV1 might be involved in change in energy substrate utilization by AITC. To elucidate whether AITC directly activates TRPV1, we examined whether AITC affected [Ca²⁺]_i in HEK293 cells expressing TRPV1. To perform this experiment, we established doxycycline-inducible cell lines expressing TRPV1. Expression of TRPV1 in these cells was confirmed by their responses to capsaicin. AITC at concentrations > 20 μM caused a significant increase of [Ca²⁺]_i in cells expressing TRPV1. AITC had no effect on cells that were not induced to express TRPV1 by doxycycline. To determine whether AITC induced the increase of [Ca²⁺]_i due to Ca²⁺ influx, the effects of extracellular Ca²⁺ ion depletion were examined. The AITC-induced increase of [Ca²⁺]_i was not observed in the absence of extracellular Ca²⁺, indicating that AITC caused Ca²⁺ influx. The peaks of [Ca²⁺]_i change induced by various concentrations of AITC were plotted and analyzed. AITC induced Ca²⁺ influx in a dose-dependent manner (Fig. 6A). TRPV1 antagonist capsazepine significantly decreased Ca²⁺ influx induced by AITC (Fig. 6B).

AITC activates capsaicin-sensitive DRG neurons from TRPA1 KO mice.

TRPV1 is expressed in a part of DRG neurons. To test whether AITC activates TRPV1-expressing but not TRPA1-expressing DRG neurons, we examined [Ca²⁺]_i measurement of DRG neuron from TRPA1 KO mice (Fig. 7, A and B). We used capsaicin to identify TRPV1-expressing sensory neurons. AITC gradually increased [Ca²⁺]_i in a subset of DRG neurons from TRPA1 KO mice (29.8%, Fig. 7B). Most of the neurons that responded to AITC also responded to capsaicin (84.4% of neurons that responded to AITC, Fig. 7B). Considering that capsaicin is a TRPV1 agonist, these results suggest that AITC activates TRPV1-expressing sensory neurons in vivo. In a subset of DRG neurons from WT mice, AITC rapidly increased [Ca²⁺]_i (Fig. 7C), however, these responses were not observed in DRG neurons from TRPA1 KO mice (for 322 DRG neurons from TRPA1 KO mice). Therefore, it was considered that the rapid response to AITC was induced via TRPA1. Consequently, AITC could activate TRPV1-expressing sensory neurons not via TRPA1.

Contribution of TRPV1 to changes in energy substrate utilization.

To examine the contribution of TRPV1 to changes in energy substrate utilization induced by AITC administration, we administered AITC to TRPV1 KO mice. In TRPV1 KO mice, intragastric administration of AITC did not elevate RER; this finding differed from that observed in WT mice (Fig. 8). RER seemed to slightly elevate in the AITC-treated group compared with that in the vehicle-treated group; however, there was no significant difference between the groups in the average RER for 2 h after administration (Fig. 9). AITC slightly increased carbohydrate oxidation and decreased fat oxidation for 2 h after administration compared with vehicle; however, there was no significant difference between the groups in the cumulative total carbohydrate oxidation and fat oxidation for 2 h after administration. V̇o₂ was not affected for at least 2 h after administration.

Investigation of the site of action of AITC for changes in energy substrate utilization.

To elucidate the site of action of AITC, we administered AITC to mice intraperitoneally and used vagotomized mice. Intraperitoneal administration of AITC elevated RER for 20–100 min after administration compared with vehicle. However, V̇o₂ was decreased for 60 min after administration in the AITC-treated group than in the vehicle-treated group (Fig. 10). Carbohydrate oxidation was slightly increased and fat oxidation was decreased for 2 h after administration of AITC in the AITC-treated group compared with that in the vehicle-treated group (data not shown).

In vagotomized mice, intragastric administration of AITC significantly elevated RER for 30–40 min after administration compared with vehicle administration. V̇o₂ was slightly higher ∼30 min after administration in the AITC-treated group than in the vehicle-treated group (Fig. 11). However, there was no significant difference between each group in the cumulative total V̇o₂ for 2 h after administration (data not shown). In sham-operated mice, intragastric administration of AITC significantly elevated RER for 20–90 min after administration compared with vehicle administration (Fig. 11). V̇o₂ was slightly higher around 30 min after administration in the AITC-treated group than in the vehicle-treated group. However, there was no significant difference between each group in the cumulative total V̇o₂ for 2 h after administration (data not shown).

DISCUSSION

In the present study, we observed that intragastric administration of AITC elevated RER, markedly increased carbohydrate oxidation, and decreased fat oxidation. V̇o₂ was slightly increased for 30 min after AITC administration; however, there was no significant difference between the AITC-treated group and vehicle-treated groups in cumulative total V̇o₂ for 2 h after administration. Therefore, AITC did not considerably affect V̇o₂.

Intraperitoneal administration of AITC elevated RER. However, V̇o₂ was decreased after AITC administration. It is considered that the dose of AITC was too high to administrate intraperitoneally and that activities of mice were suppressed by damages from AITC. In previous studies, we showed that intragastric administration of AITC to anesthetized mice increased colon and interscapular brown adipose tissue temperatures and decreased tail temperature (36). We also obtained similar results in the experiments of intravenous administration of AITC (unpublished data). These reports suggest the possibility that AITC acts not only in the preabsorptive state but also in the postabsorptive state through the bloodstream and affects carbohydrate oxidation.

In vagotomized mice, AITC also elevated RER similar to sham-operated mice. This result indicated that vagus nerves were not involved in an increase in carbohydrate oxidation by AITC. If AITC acts within gastrointestinal tracts, extrinsic nerves are involved in transmission of stimulation by AITC. Extrinsic nerves of the gastrointestinal tract are vagus and spinal nerves and the literature contains reports of vagal and spinal afferent fibers expressing TRPA1 and V1 (49, 61). In the present study, our data indicate that vagus nerves are not involved in changes of whole body metabolisms by AITC. Together with intraperitoneal administration experiments, the action site of AITC might be beyond the gastrointestinal tract or spinal nerves within the gastrointestinal tract. It is important to elucidate the action site of AITC, and it needs further studies.

The mechanisms by which AITC causes an increase in carbohydrate oxidation remain unclear. At present, we demonstrate that TRPV1 was involved in increase in carbohydrate oxidation by AITC. Since it has been reported that TRPV1 is expressed in primary sensory neurons and DRG (49, 50, 61), AITC might increase carbohydrate oxidation via TRPV1 expressed in the sensory nerve terminals of the gastrointestinal tract or throughout the body. In fact, capsaicin, a TRPV1 agonist, induced adrenaline secretion primarily through activation of the adrenal sympathetic nerve (58). Furthermore, it is reported that TRPV1 is involved in insulin secretion and glucose metabolism (1, 15–17). It was assumed that adrenaline secretion through activation of the central nervous system was induced by AITC and that glucose uptake was increased via β-adrenoreceptor activation by noradrenaline in peripheral tissues, skeletal muscles, and brown adipose tissues (42). Further studies are required to elucidate where and how carbohydrate is metabolized.

Intragastric administration of the TRPV1 agonists capsaicin and capsiate did not increase carbohydrate oxidation similar to AITC (28). However, intraperitoneal administration of capsaicin to anesthetized rats elevated RER (30). Capsaicin is absorbed from the gastrointestinal tract and is to a great extent metabolized in the liver before it reaches the general circulation (11, 29). AITC is also absorbed from the gastrointestinal tract, and intact AITC is detected in urine (6, 19, 34). Consequently, differences in stability and action site of these compounds may lead to differences in metabolic changes of carbohydrate oxidation.

It is possible that differences in binding sites of AITC and capsaicin in TRPV1 caused a differential effect on carbohydrate oxidation. It has been reported that TRPV1 is activated by multiple pathways and has multiple ligand-binding sites (56). For example, not one but three amino acids are involved in TRPV1 activation by capsaicin and other vanilloids, and some other amino acids are involved in TRPV1 activation by proton, one of the TRPV1 agonists(14, 25, 27, 39). Since AITC is not a vanilloid, its binding site for TRPV1 activation may be different from that of capsaicin. A recent study suggested that a single NH₂-terminal cysteine plays an important role in the activation of TRPV1 by allicin (48). AITC is structurally similar to allicin; therefore, TRPV1 activation by AITC may use the same binding site as TRPV1 activation by allicin. Further studies are needed to elucidate this point.

Intragastric administration of capsaicin at high doses (20 or 50 mg/kg) did not elicit an increase in carbohydrate oxidation similar to AITC (data not shown). Therefore, it is suspected that not only pain stress but also other mechanisms may be involved in the increase of carbohydrate oxidation by AITC.

Intragastric administration of cinnamaldehyde, a TRPA1 agonist, did not increase carbohydrate oxidation. The dose of cinnamaldehyde was determined from a previous in vitro study (4). In that study, the concentration for half-maximal activation of TRPA1 (EC₅₀ value) by AITC was three times greater than cinnamaldehyde (22 and 61 mM, respectively). In this study, we used cinnamaldehyde at the dose of three times greater than the effective dose of AITC to increase carbohydrate oxidation (760 μmol/kg and 250 μmol/kg, respectively). If intragastric administration of AITC activated TRPA1 in the present experiment, the dose of cinnamaldehyde was thought to be sufficient to activate TRPA1. However, we observed that cinnamaldehyde did not increase carbohydrate oxidation at that dose. Moreover, AITC increased carbohydrate oxidation in TRPA1 KO mice. Consequently, it is considered that TRPA1 is not involved in the increase in carbohydrate oxidation by AITC.

AITC did not increase carbohydrate oxidation in TRPV1 KO mice, suggesting that the increase in carbohydrate oxidation by AITC observed in this study is mediated by TRPV1. Furthermore, we demonstrated that AITC dose-dependently increased [Ca²⁺]_i in cells expressing TRPV1. An AITC-induced increase of [Ca²⁺]_i was not observed in the absence of extracellular Ca²⁺, indicating that AITC caused Ca²⁺ influx. Since TRPV1 is a Ca²⁺-highly permeable nonselective cation channel (8), it is considered that AITC increased [Ca²⁺]_i via activation of TRPV1. In previous studies, AITC did not activate TRPV1 (24, 48). In the present study, we used different experimental methods from those of previous studies, which may have resulted in obtaining different results. Compared with the previously reported EC₅₀ value (711 nM or 1.47 μM) of representative TRPV1 agonist capsaicin (8, 33), the EC₅₀ (∼200 μM) of AITC was considerably high. At high doses, it is likely that AITC exerts a nonspecific effect on HEK293 cells. However, AITC did not increase [Ca²⁺]_i in cells in which TRPV1 was not induced by doxycycline and addition of the TRPV1 antagonist capsazepine decreased Ca²⁺ influx by AITC; therefore, AITC increased [Ca²⁺]_i in cells via TRPV1.

AITC increased [Ca²⁺]_i in a subset of DRG neuron from TRPA1 KO mice. There is no difference between the number of AITC responding neurons and capsaicin responding neurons in the DRG neurons (by χ² tests). Most of the neurons that responded to AITC also responded to capsaicin. Together with HEK293 cell data, we considered that AITC could directly activate TRPV1 in vivo. However, some points have remained unclear (eg., differences between time course of [Ca²⁺]_i by AITC and capsaicin). To elucidate these points, more detailed studies are needed in the future.

A previous study of Akopian (2) reported that trigeminal ganglion (TG) neurons from TRPA1 KO mice were not responsive to AITC. In this study, we used not TG neurons but DRG neurons. The application time of AITC for TG neurons was only 2 min, but that for DRG neurons, in this study, was more than 10 min. In the present study, the response to AITC in DRG neurons from TRPA1 KO mice was slow compared with that of capsaicin. Considering these facts, the application time of AITC in the previous study might be too short to observe the response to AITC in neurons from TRPA1 KO mice. In fact, we observed the rapid response to AITC (that was within 2 min) in neurons from WT mice and not in TRPA1 KO mice. Differences between the methodologies of previous reports and that of our study might explain these unexpected results.

It has been reported that TRPA1 is coexpressed with TRPV1 in a subset of small-to-medium diameter peripheral sensory neurons (24, 52). In the present study, we demonstrated that AITC activated TRPV1. From these reports and our present data, it was expected that cross-interaction between TRPA1 and TRPV1 (47) might be involved in the changes in metabolisms by AITC. However, TRPA1 is not involved in the increase in carbohydrate oxidation by AITC because AITC increased carbohydrate oxidation in TRPA1 KO mice similar to WT mice. It is, therefore, unlikely that cross-interaction is involved in the changes in metabolisms by AITC in the present study.

In the present study, AITC directly activated TRPV1 in vitro experiments. However, it is possible that TRPV1 was indirectly activated by AITC in vivo. It is reported that bradykinin, ATP, prostaglandins, and trypsin or tryptase are inflammatory mediators involved in the activation of TRPV1 by protein kinase C-dependent phosphorylation (9, 40, 53, 57). AITC might induce inflammation, resulting in the release of bradykinin, ATP, prostaglandins, and trypsin or tryptase, which might affect TRPV1 activation.

It is possible that the effects of AITC on the increase in carbohydrate oxidation were caused by metabolites. It has been reported that AITC was metabolized to some compounds after oral administration (19). We showed that intraperitoneal administration of AITC also elevated RER, indicating that AITC could act without gastric digestion. Although it could not be denied completely that metabolites of AITC are involved in the increase in carbohydrate oxidation by AITC, we considered that AITC is important to the increase in carbohydrate oxidation by AITC.

We administered AITC to mice in high doses. Concentrations of AITC solution administered to mice were 50 and 100 mM, which is too high to get from spicy meals. In previous studies, absorption and metabolism of AITC were different between rats and mice (6, 19), indicating that there might be species differences in effective concentration of AITC. So we consider that more lower concentration or long-term administration at low concentration might be effective in application for humans. Further studies are required to consider application for humans.

Recent studies have indicated the evidence to support the concept of TRP channels as targets in metabolism (3, 22, 28, 36, 44, 51). In those reports, activation of TRPV1 and TRPA1 was considered to cause reflex activation of sympathetic pathways or inactivation of parasympathetic pathways via the central nervous system (23, 30, 32, 58, 59). Other reports indicate a relationship between TRPV1 and blood glucose metabolism (1, 15–17). TRPV1 and TRPA1 are also involved in nociception. It might be reasonable to consider that changes in metabolisms through the central nervous system are involved in responses to avoid painful stimuli. Consequently, it is expected that activation of TRPV1 or TRPA1 affect energy metabolism, and in future they would be important targets in the modulation of energy metabolisms.

In conclusion, we demonstrated that intragastric administration of AITC increased carbohydrate oxidation in mice and that the effects were mediated by TRPV1 but not TRPA1. We also showed that AITC increased [Ca²⁺]_i in cultured cells and in DRG neurons and suggested that AITC might directly activate TRPV1.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).