Am J Physiol Regul Integr Comp Physiol 295: R82–R91, 2008.
First published May 14, 2008; doi:10.1152/ajpregu.00060.2008.
Mercaptoacetate inhibition of fatty acid ␤-oxidation attenuates the oral
acceptance of fat in BALB/c mice
Shigenobu Matsumura, Katsuyoshi Saitou, Takashi Miyaki, Takeshi Yoneda, Takafumi Mizushige,
Ai Eguchi, Tetsuro Shibakusa, Yasuko Manabe, Satoshi Tsuzuki, Kazuo Inoue, and Tohru Fushiki
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Submitted 27 January 2008; accepted in final form 12 May 2008
licking behavior; fat; palatability
and generally more palatable to
humans than low-calorie, low-fat foods. This preference is also
observed in mice and rats (6, 29, 33). Yoneda and colleagues
(47, 48) demonstrated that mice prefer a higher concentration
of corn oil to a lower one in both a short-term licking test and
a two-bottle choice test; moreover, the reinforcing effect of
corn oil was enhanced in proportion to the concentration of
corn oil in an operant lever-press task.
Degrees of sweetness, sourness, and saltiness can easily be
discriminated in the oral cavity. But humans are unable to
recognize pure fat as a taste. And, although fat content in foods
is an important factor in their palatability, there is very little
information about how fat concentrations in foods are sensed.
Among the limited number of studies that have been reported
on this subject, several have indicated that rodents and humans
recognize the presence of fat in foods not only by texture but
also chemically in the mouth (10, 11, 14, 40). CD36 and
GPR120, which are expressed in the taste bud cells, are
candidates for fatty acid receptors in the oral cavity (9, 22, 37).
Generally, energy content in foods is evaluated in the postingestive process, which includes the secretion of gastrointestinal
hormones, activation of the vagus nerve, elevation of the blood
levels of energy substrates, etc. In addition, several tissues, including the brain, pancreas, and liver, monitor energy substrate
utilization in terms of ATP production (20, 25, 26, 28).
FAT IS AN ATTRACTIVE FOOD,
Address for reprint requests and other correspondence: S. Matsumura,
Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto Univ., Oiwakecho, Kitashirakawa,
Sakyo, Kyoto, Japan 606-8502 (e-mail: [email protected]).
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Several studies have reported that peripheral fat metabolism
affects feeding behavior (5, 36, 39). In studies on the energy
metabolism of fat, mercaptoacetate is widely used to inhibit the
␤-oxidation of fatty acid chains by attenuating acyl-CoA dehydrogenase activity (3). In a previous study using the conditioned place preference (CPP) test, we demonstrated that intraperitoneal mercaptoacetate administration abolishes the reinforcing effect of corn oil (fat) in mice (42). Although sucrose
solution also had a reinforcing effect in the CPP test, this effect
was not inhibited by mercaptoacetate, presumably because the
metabolism of sucrose differs from that of corn oil. Thus
mercaptoacetate does not exert general effects but specifically
suppresses the reinforcing effects of fat. In macronutrient
self-selection studies, Singer et al. (38) and Ritter et al. (34)
found that mercaptoacetate increased protein and carbohydrate
ingestion and decreased fat ingestion, suggesting that metabolic change accompanied by reduced energy production from
fat affects the pattern of feeding behavior.
Furthermore, the mercaptoacetate-induced increase in food
intake is abolished by subdiaphragmatic or hepatic branch
vagotomy (21, 35), and intraportal mercaptoacetate injection
increases the discharge rate in hepatic vagal afferents (24).
Methyl palmoxirate and etomoxir, both known to be carnitine
palmitoyl transferase-I inhibitors, increase food intake by decreasing the ATP-to-ADP ratio in the liver (15, 19). These
results clearly suggest that the peripheral tissues innervated by
the vagus nerve have a system for monitoring fat metabolism
and/or fat utilization in the postingestive process. However, it
appears certain that there is a system for evaluating fat as
energy in the oral cavity because animals can recognize fat
within a few seconds and show a high affinity for it.
In the present study, we focused on the mechanism in the
oral cavity for recognition of fat as an energy source. We
examined this mechanism by using mercaptoacetate to inhibit
mitochondrial fatty acid ␤-oxidation.
MATERIALS AND METHODS
Animals. Eight-week-old male BALB/c mice were obtained from
Japan SLC (Hamamatsu, Japan) for each experiment. The mice were
housed individually in a vivarium maintained at 23 ⫾ 2°C under a
12:12-h light-dark cycle (lights on from 6:00 –18:00 h). A commercial
standard laboratory chow (MF; Oriental Yeast, Tokyo, Japan) and water
were available ad libitum. The caloric ratios of protein, fat, and carbohydrates in the chow were 26.2%, 13.3%, and 60.5%, respectively. The
mice were maintained for 1 wk after arrival to permit them to acclimatize
to their surroundings before being tested. All experiments were carried
out during the daytime (10:00 –18:00 h). This study was conducted in
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0363-6119/08 $8.00 Copyright © 2008 the American Physiological Society
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Matsumura S, Saitou K, Miyaki T, Yoneda T, Mizushige T,
Eguchi A, Shibakusa T, Manabe Y, Tsuzuki S, Inoue K, Fushiki
T. Mercaptoacetate inhibition of fatty acid ␤-oxidation attenuates the
oral acceptance of fat in BALB/c mice. Am J Physiol Regul Integr
Comp Physiol 295: R82–R91, 2008. First published May 14, 2008;
doi:10.1152/ajpregu.00060.2008.—We investigated the effect of
␤-oxidation inhibition on the fat ingestive behavior of BALB/c mice.
Intraperitoneal administration to mice of mercaptoacetate, an inhibitor of
fatty acid oxidation, significantly suppressed intake of corn oil but not
intake of sucrose solution or laboratory chow. To further examine the
effect of mercaptoacetate on the acceptability of corn oil in the oral
cavity, we examined short-term licking behavior. Mercaptoacetate significantly and specifically decreased the number of licks of
corn oil within a 60-s period but did not affect those of a sucrose solution,
a monosodium glutamate solution, or mineral oil. In contrast, the administration of 2-deoxyglucose, an inhibitor of glucose metabolism, did not
affect the intake or short-term licking counts of any of the tasted
solutions. These findings suggest that fat metabolism is involved in the
mechanism underlying the oral acceptance of fat as an energy source.
MERCAPTOACETATE ATTENUATES ACCEPTANCE OF FAT
accordance with the ethical guidelines of the Kyoto University Animal
Experimentation Committee and was in complete compliance with the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals. All procedures were approved by the Kyoto University Animal
Care and Use Committee. All efforts were made to minimize the number
of animals used and limit experimentation to that which was necessary to
produce reliable scientific information.
Respiratory gas analysis. The mice were held individually in a
chamber for 12 h so that they could attain a constant respiratory
exchange ratio (RER). Then either mercaptoacetate (Wako Pure
Chemical Industries, Osaka, Japan; n ⫽ 5– 6) or saline (control; n ⫽
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8) was intraperitoneally administered, and the expired air was analyzed. The oxidation of total fatty acids and carbohydrates was
computed based on oxygen consumption (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 (17, 18). 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.
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Fig. 1. A: effect of the intraperitoneal administration of
mercaptoacetate (MA) or 2-deoxyglucose (2-DG) on exogenous substrate oxidation. MA or 2-DG was administered
intraperitoneally 30 min before the intragastric infusion of
13
C-labeled oleic acid or 13C-labeled glucose. Values are
means ⫾ SE. n ⫽ 5– 8 (saline vs. MA 400 ␮g/kg: P ⬍ 0.01
at 30 –120 min; saline vs. MA 600 ␮mol/kg: P ⬍ 0.01 at
30 –120 min; saline vs. 2-DG 200 mg/kg: P ⬍ 0.01 at 30 – 60
min; saline vs. 2-DG 400 mg/kg: P ⬍ 0.01 at 30 – 60 min;
2-way repeated ANOVA, followed by Bonferroni’s post hoc
test). B: changes in the oxygen consumption of mice administered MA or 2-DG after intragastric infusion. Values are
means ⫾ SE. n ⫽ 5– 8 (saline vs. 2-DG 200 mg/kg: P ⬍
0.01 at 0 –5 min; saline vs. 2-DG 400 mg/kg: P ⬍ 0.01 at 0 –5
min; 2-way repeated ANOVA, followed by Bonferroni’s multiple comparison test as a post hoc test). C: changes in the
respiratory exchange ratio of mice administered MA or
2-DG after intragastric infusion. Values are means ⫾ SE.
D: cumulative substrate oxidation of mice administered MA
or 2-DG after intragastric infusion. FAT, total fatty acid;
CHO, carbohydrates. Values are means ⫾ SE. n ⫽ 5– 8;
*P ⬍ 0.05 (Tukey’s test).
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MERCAPTOACETATE ATTENUATES ACCEPTANCE OF FAT
which it was recessed ⬃5 mm to restrict contact with the paws, nose,
jaw, and other body parts. Licking rates were converted from the
positive voltage peak every 10 s.
On day 1, the mice were placed in the stainless steel test cage for 30
min without any fluids to allow them to become habituated to the cage.
On day 2, the mice were given a solution to sample. On the test day (day
3), the mice received intraperitoneal mercaptoacetate (n ⫽ 12), 2-deoxyglucose (n ⫽ 12) or saline (n ⫽ 12) and were then placed in the test cage.
Thirty minutes after administration, the mice were offered the same
sample solution as on day 2. Licking rates were calculated for 60 s from
the first lick, and fluid intake was also measured for 30 min.
Statistical analysis. All values are presented as means ⫾ SE. The
effects of the administration of mercaptoacetate or 2-deoxyglucose on
exogenously administered substrate oxidation, RER, and oxygen consumption were examined by two-way repeated-measures ANOVA
with the Bonferroni post hoc test (Prism 4.0; GraphPad Software, San
Diego, CA). The effects of the administration of mercaptoacetate or
2-deoxyglucose on fluid intake were examined by one-way ANOVA
(see Figs. 3 and 7). Tukey’s test was used as a post hoc test. The
effects of the administration of mercaptoacetate or 2-deoxyglucose on
ingestive behavior (see Figs. 2, 4 – 6, 9, and 10) were examined using
an unpaired t-test.
RESULTS
Effects of mercaptoacetate and 2-deoxyglucose on exogenously administered substrate oxidation. In the control group
(saline ip), the oral ingestion of 13C-labeled oleic acid rapidly
Licking test
In the licking test, mice that had ingested corn oil at least three
times were used. The test was carried out in a handmade licking test
chamber. The principle and apparatus were similar to those described
by Hayar’s method (13). Briefly, the mice were placed in a stainless
steel cage (32 cm ⫻ 22 cm ⫻ 10.5 cm) connected to a standard
analog-to-digital converter (Mac Lab/8S; ADInstrument, Castle Hill,
NSW, Australia) through two cables. One cable was attached to a
stainless steel drinking spout (3.5-cm long with an 8-mm orifice; the
spout had an internally located stainless steel ball) and the other was
attached to the cage. The spout was protected by a silicon sleeve into
Fig. 2. Cumulative food intake after the intraperitoneal administration of MA
(600 ␮mol/kg) or 2-DG (200 mg/kg). Values are means ⫾ SE. n ⫽ 8; *P ⬍
0.05; **P ⬍ 0.01 (Student’s t-test).
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Based on the volume of CO2 production per unit of time (l/min;
V̇CO2) and V̇O2, the total glucose and lipid oxidation were calculated
using the stoichiometric equations of Frayn (7) as follows: total fatty
acid oxidation ⫽ 1.67 V̇CO2 ⫺ 1.67 V̇O2, and carbohydrate oxidation ⫽ 4.55 V̇O2 ⫺ 3.21 V̇CO2.
Oxidation of exogenous fat was assessed based on the relative
abundance of 13CO2 in respiratory gas after intragastric ingestion of
13
C-labeled oleic acid. 13C-labeled oleic acid was added in quantities
calculated to result in a concentration similar to that recommended by
Ishihara et al. (17) as follows. A solution containing 0.3% methylcellulose (emulsifier), 10% corn oil (Wako), and 0.02 mol/l of 13Clabeled oleic acid (Isotec, Tokyo, Japan) was administered to mice
intragastrically (0.01 ml/g of body mass) 30 min after intraperitoneal
mercaptoacetate administration. The total amount of 13C-labeled oleic
acid administered was 0.2 mmol/kg of body mass.
In the case of 2-deoxyglucose (Wako) administration, a solution
containing 0.3% methylcellulose, 10% glucose, and 0.02 mol/l of
13
C-labeled glucose (Isotec) was administered to mice intragastrically
(0.01 ml/g of body mass) 30 min after intraperitoneal 2-deoxyglucose
administration (n ⫽ 5– 8). The total amount of 13C-glucose administered was 0.2 mmol/kg of body mass.
Measurement of food intake. Food intake was measured 4 h after
the lights went on (10:00 h). The mice were reared individually and
were allowed food and water ad libitum during the experiment.
Mercaptoacetate (600 ␮mol/kg; n ⫽ 8), 2-deoxyglucose (200 mg/kg;
n ⫽ 8), and saline (n ⫽ 8) were administered intraperitoneally. Food
intake was measured for 60-, 120-, and 240-min periods. Spilled food
was weighed and food intake was corrected as necessary.
Mearsurements of fluid intake. All mice were reared individually,
and all training and tests were performed individually. During training, the mice were given access to corn oil in their home cages for 10
min every other day. During each training session, the mice ingested
corn oil at least three times; each time food and water were removed
30 min prior to the access to corn oil. In the test session, the mice
received intraperitoneal mercaptoacetate (n ⫽ 8), 2-deoxyglucose
(n ⫽ 8), or saline (control: n ⫽ 8) at the same time that food and water
were removed. Thirty minutes later, the mice were offered corn oil.
The intake of corn oil over 10- and 120-min periods was measured. In
the case of sucrose solution, 2.5%, 10%, 20%, and 40% solutions were
used, and fluid intake was measured by the same procedure as used in
the corn oil tests. The intakes of olive oil (Wako), soybean oil (Wako),
safflower oil (ICN Chemicals, Costa Mesa, CA), triolein (SigmaAldrich, St. Louis, MO), and Intralipid (Terumo, Tokyo, Japan) were
measured by the same procedure as used in the corn oil test.
To examine the effect of mercaptoacetate administration on corn
oil ingestion, only mice that had never ingested corn oil before were
used. On the first day (day 1) and day 8, the mice were intraperitoneally
administered mercaptoacetate (600 ␮mol/kg: n ⫽ 8) or saline (n ⫽ 8),
and food and water were immediately removed. Thirty minutes later, the
mice were offered corn oil for 10 min and the intake of corn oil was
measured in each mouse. From day 1 to day 10, the mice were offered
corn oil for 10 min every other day, and their intake of oil during each test
period was recorded by the same procedure used on day 1 but without any
mercaptoacetate administration.
MERCAPTOACETATE ATTENUATES ACCEPTANCE OF FAT
Effects of mercaptoacetate and 2-deoxyglucose on food intake.
The intraperitoneal mercaptoacetate administration (600 ␮mol/
kg) slightly increased food intake over the 60- and 120-min
periods, but there were no significant differences between the
mercaptoacetate-administered group and the saline-administered group (Fig. 2).
The intraperitoneal administration of 2-deoxyglucose (200
mg/kg) significantly increased cumulative food intake in the
60-, 120-, and 240-min periods compared with the salineadministered group.
Effects of mercaptoacetate on fluid intake. Intraperitoneal
mercaptoacetate administration dose-dependently decreased
corn oil intake during the short-term tests (10- and 120-min
periods) compared with the saline-administered group (Fig. 3).
Furthermore, mercaptoacetate decreased the intakes of olive
oil, soybean oil, safflower oil, and triolein (Fig. 4); however, it
did not affect the intake of a 2.5– 40% sucrose solution (Fig. 3).
Mercaptoacetate significantly decreased the intake of fat
emulsion (Intralipid: 20% soybean oil) (Fig. 5). However, the
addition of sucrose (10%, 20%, 40%) to Intralipid dose-dependently increased the intake of Intralipid in the mercaptoacetateadministered group. No significant differences were observed
between the mercaptoacetate- and saline-administered groups in
the intake of Intralipid with the addition of 40% sucrose.
Effects of mercaptoacetate on daily changes in corn oil
intake. To test whether mercaptoacetate administration causes an
aversive response to corn oil, we measured the daily corn oil
intake of mercaptoacetate-administered mice that had never ingested corn oil before. Mice of both the mercaptoacetate group
and the control group consumed little corn oil on day 1 because
Fig. 3. Fluid intake after the intraperitoneal administration of mercaptoacetate. Values are means ⫾ SE.
n ⫽ 8; *P ⬍ 0.05; **P ⬍ 0.01 (Tukey’s test).
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increased the 13CO2-to-12CO2 ratio in expired air, perhaps as a
result of the oxidization of 13C-labeled oleic acids (Fig. 1).
Intraperitoneal mercaptoacetate administration completely suppressed the increase in the 13CO2-to-12CO2 ratio in expired air
at a dose of 400 ␮mol/kg or of 600 ␮mol/kg. Mercaptoacetate
did not affect oxygen consumption (V̇O2) for at least 2 h after
administration (Fig. 1). RER was slightly higher in the mercaptoacetate-administered group than in the saline-administered group, and cumulative fatty acid oxidation over 60 to 120
min tended to be lower. These results suggest that a single
intraperitoneal administration of mercaptoacetate strongly inhibits exogenous fat oxidation and moderately inhibits endogenous fat oxidation. Since exogenous fat oxidation was inhibited for at least 30 to 90 min after administration, we chose 30
min after mercaptoacetate administration as the time to evaluate ingestive behavior.
The intraperitoneal administration of 2-deoxyglucose dosedependently suppressed exogenously administered carbohydrate
(glucose) oxidation, and significant differences were observed at
a dose of 200 mg/kg. A dose of 400 mg/kg 2-deoxyglucose
completely suppressed exogenously administered carbohydrate
oxidation. Both the 200 mg/kg and the 400 mg/kg doses of
2-deoxyglucose significantly decreased oxygen consumption
immediately after administration. A dose of 400 mg/kg 2-deoxyglucose caused depression and significantly attenuated
spontaneous motor activity, probably due to severe glucoprivation. Thus, a dose of 200 mg/kg 2-deoxyglucose was used in
further experiments. None of the doses of 2-deoxyglucose
affected RER, but all moderately decreased the cumulative
total carbohydrate oxidation.
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mice are neophobic to unfamiliar foods. In the saline-administered
group, however, intake increased daily beginning on the second
ingestion (day 2) and continuing until at least day 5 (Fig. 6).
Although mice were not administered mercaptoacetate on day 2,
no increase in intake was observed in the mercaptoacetate-administered group on that day. On day 8, similar to the pattern shown
in Fig. 3, intake decreased in the mercaptoacetate group, but
mercaptoacetate did not affect intake on the following day (day 9)
and did not cause an aversive response to corn oil.
Effects of 2-deoxyglucose on fluid intake. The intraperitoneal
administration of 2-deoxyglucose did not affect the intake of
corn oil (10 min or 120 min) (Fig. 7); nor did it affect the intake
of any concentration (2.5– 40%) of sucrose solution.
Effects of mercaptoacetate and 2-deoxyglucose on licking
behavior for fat, sucrose, and monosodium glutamate solution
(lickometer analysis). We used a licking test to rule out the
possibility that postingestive feedback of ingested fat affected
the palatability of fat. Mercaptoacetate (600 ␮mol/kg) significantly suppressed the number of licks of corn oil during the
60-s period beginning with the first lick (initial licking rate;
Figs. 8 and 9), but did not affect the rate of sucrose solution
Fig. 5. Intake of Intralipid with the addition of various concentrations of
sucrose after the intraperitoneal administration of mercaptoacetate (600 ␮mol/
kg). Values are means ⫾ SE. n ⫽ 6 – 8; *P ⬍ 0.01 (Student’s t-test).
Fig. 6. Changes in the daily intake of corn oil. Mice were administered
mercaptoacetate (600 ␮mol/kg) or saline on day 1 and day 15. Values are
means ⫾ SE. n ⫽ 8; *P ⬍ 0.01 (Student’s t-test).
licking. Mercaptoacetate did not affect the licking rate for a 0.1
M monosodium glutamate solution or for mineral oil (Fig. 9).
There were no differences in the interval between fluid presentation and the onset of the first lick (latency) between the
mercaptoacetate-administered group and the saline-administered group (data not shown). 2-Deoxyglucose did not affect
the number of licks of corn oil or sucrose solution (Fig. 10).
Latency was not affected by 2-deoxyglucose (data not shown).
DISCUSSION
In the present study, we demonstrated that the intraperitoneal administration of mercaptoacetate markedly suppressed
the short-term licking behavior of mice, which is thought to be
a good index of affinity for a test solution (4). The suppression
of licking behavior by mercaptoacetate was highly fat specific
and was not observed at all when either a sweet (sucrose)
solution or an umami (monosodium glutamate) solution was
presented.
We should pay special attention to the fact that the decrease
in the licking rate was manifested within several seconds after
mercaptoacetate administration. Given this very short period,
we can rule out any contribution of postingestive feedback to
licking behavior. However, it remains unclear how mercaptoacetate induced such a dramatic change in ingestive behavior in
such a short period. Previous studies have focused primarily on
the effect of mercaptoacetate on the postingestive process of fat
metabolism. For example, it has been reported that vagotomy
attenuates the ability of mercaptoacetate to elicit feeding behavior (12, 35). In our laboratory, Suzuki (42) showed that
mercaptoacetate abolishes the reinforcing effect of corn oil,
suggesting that mercaptoacetate influences the postingestive
effect of fat. Ackroff and colleagues (1, 2) also showed that
the postingestive energy signal plays an important role in the
maintenance of high palatability. Although mercaptoacetate
influences the postingestive effect of fat, the present data
suggest that mercaptoacetate also influences the recognition of
fat in the oral cavity, raising the possibility that the energy
content of fat is evaluated in the oral cavity before the energy
signal emerges during the postingestive process.
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Fig. 4. Intake of various fats after the intraperitoneal administration of MA
(600 ␮mol/kg). Values are means ⫾ SE. n ⫽ 8; *P ⬍ 0.01 (Student’s t-test).
MERCAPTOACETATE ATTENUATES ACCEPTANCE OF FAT
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Fig. 8. Typical patterns of the licking behavior of mercaptoacetate- and
saline-administered mice. Licking was recorded beginning with the first lick.
Each spike indicates one lick.
Mercaptoacetate is widely used to inhibit the ␤-oxidation of
fatty acid chains by attenuating acyl-CoA dehydrogenase activity. In the present study, we demonstrated that mercaptoacetate completely suppressed the oxidation of exogenously
administered fat. In contrast, it only modestly suppressed
endogenous fat oxidation. It remains unclear why the suppression of fat oxidation by mercaptoacetate was different between
exogenous and endogenous fat. This finding may have been
due to our use of indirect calorimetry, which is a measure of
the whole-body metabolism. The absolute value of exogenous
fat oxidation is smaller than that of whole-body (endogenous)
fat oxidation and exogenously administered fat is oxidized only
in specific tissues. Therefore, we were unable to detect any
massive changes in endogenous (whole-body) fat oxidation.
Additionally, there are several differences between endogenous fat and exogenous fat in terms of the degradation process,
the form of transport and the metabolic mechanism.
Using a two-bottle choice test, we previously demonstrated
that mice exhibit a high affinity for corn oil (47). Other studies
have used the CPP test, which evaluates the reinforcing effects
of additive drugs and food rewards (23, 32, 41, 44) and demonstrated that corn oil ingested by mice for three consecutive days
had a reinforcing effect (16). Mizushige and colleagues (30,
31) also observed that daily repetitive ingestion of corn oil
enhances the motivation to ingest corn oil and increases the
expression level of a ␤-endorphin precursor, proopiomelanocortin mRNA, in the hypothalamus. ␤-Endorphin is known to
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Fig. 7. Fluid intake after the intraperitoneal administration of 2-DG. Values are means ⫾ SE. n ⫽ 8.
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Fig. 9. Effect of mercaptoacetate on licking behavior. A: cumulative licking counts for corn oil and sucrose solution in mice
administered mercaptoacetate (600 ␮mol/kg). Values are
means ⫾ SE. n ⫽ 12. B: initial licking rate. Values are means ⫾
SE. n ⫽ 12; *P ⬍ 0.01 (Student’s t-test).
be an endogenous opioid peptide involved in the reward
system. Therefore, it is presumed that the repetitive ingestion
of corn oil activates the reward system and, as a result, induces
an increase in the intake of corn oil and high affinity for corn
oil. Thus, in the present study, we used mice that had ingested
corn oil at least three times for all experiments. Despite the fact
that the mice showed a high affinity for corn oil before the test
day, mercaptoacetate administration significantly decreased
corn oil intake and suppressed licking behavior for corn oil.
The licking rate for corn oil in mercaptoacetate-administered
mice corresponded to the licking rate for mineral oil, whose
texture is similar to that of corn oil. Mice prefer the oily texture
and show a certain frequency of licking. It is unlikely that
mercaptoacetate lowered olfaction or tactile sensation. Mercaptoacetate did not change the interval between fluid presentation and onset of licking (latency), nor did it affect food
intake, indicating that it did not suppress appetite or the
motivation to eat. It appears that mercaptoacetate-administered
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MERCAPTOACETATE ATTENUATES ACCEPTANCE OF FAT
mice were able to sense the oily texture but could not chemically detect the presence of fat in the oral cavity.
Mercaptoacetate did not affect the intake of the sucrose
solution at any concentration. In addition, as shown in Fig. 6,
it is clear that the mice did not show an averse response to fat
due to mercaptoacetate. Without mercaptoacetate administration, the mice favorably ingested Intralipid. Although mercaptoacetate decreased the intake of this fat emulsion, sucrose
dose-dependently increased the intake of Intralipid in the
mercaptoacetate-administered group. These results indicate
that mercaptoacetate decreases the intake of Intralipid but does
not affect the palatability of the sucrose solution added to
Intralipid. The mice showed a strong affinity for a mixed
solution of fat and sugar; these molecules synergistically enhanced the palatability of the mixed solution and increased
intake. However, mercaptoacetate administration was not followed by any such synergistic effect of fat and sugar; that is,
mercaptoacetate-administered mice did not increase their intake of the mixed solution.
To inhibit carbohydrate oxidation, we used the glucose
analog 2-deoxyglucose. The administration of 2-deoxyglucose
did not affect corn oil intake in mice. In addition, although
2-deoxyglucose induced glucoprivation and increased food
intake, it did not affect the intake of the sucrose solution at any
concentration. Furthermore, 2-deoxyglucose did not change the
initial licking rate, an index of affinity for a test solution. These
results suggest that the effect of a carbohydrate oxidation
inhibitor on feeding or ingestive behavior is not identical to
that of a fat oxidation inhibitor. Glucose is an essential energy
source and glucose homeostasis in the body is tightly regulated. For this reason, if animals are faced with fasting hypoglycemia or inhibition of carbohydrate oxidation by pharmacological manipulation, they must increase their carbohydrate
consumption. The inhibition of fat oxidation attenuates the oral
acceptance of fat, while the inhibition of carbohydrate oxidation is not thought to attenuate the oral acceptance of carbohydrate (sweet). However, in the present experiments, the dose
of 2-deoxyglucose was not sufficient to completely inhibit
exogenous carbohydrate oxidation. For this reason, we cannot
eliminate the possibility that a higher dose of 2-deoxyglucose
might affect licking behavior.
Several studies using mercaptoacetate or carnitine palmitoyl
transferase-I inhibitor have indicated that ␤-oxidation inhibition increases food intake in rats and mice (15, 36, 39). The
most prominent increase in food intake was observed in rats
fed a high-fat (⬎30%) diet (39). The present results are
somewhat different from those of other previous reports that
showed an increase in high-fat diet intake by mercaptoacetate
administration. In the studies in which mercaptoacetate induced an increase in food intake, rodents had no choice but to
eat the high-fat diet to obtain energy. In the present study, we
demonstrated that mercaptoacetate administration inhibits the
oxidation of exogenous fat; thus, mercaptoacetate-administered mice cannot acquire energy from ingested (exogenous)
fat. Hence, when the energy content of available food is
limited, mercaptoacetate-administered mice eat progressively
more of a high-fat diet to obtain adequate amounts of energy
from the carbohydrates it contains. Since we used a relatively
high-carbohydrate diet, we did not observe any increase in food
intake due to mercaptoacetate. Thus, our present and previous
results suggest that mercaptoacetate neither affects the affinity for
a high-fat diet nor stimulates the ingestion of a high-fat diet.
Several studies have demonstrated the existence of chemical
receptors for fat in the oral cavity (10, 11, 43), and CD36,
GPR40, and GPR120 have been proposed as candidates for the
fatty acid receptor in the taste bud cells on the tongue (8, 9, 22,
27). Laugerette et al. (22) and Fushiki and Kawai (9) report that
mice with a genetic deletion of CD36 are unable to recognize
fat, suggesting that mice might recognize fat in part through
CD36 expressed in the taste bud cells. It seems likely that mice
evaluate fat via fatty acid ␤-oxidation in the taste bud cells.
CD36, also known as fatty acid translocase, may be related to
this mechanism. However, to the best of our knowledge, no
direct evidence of such a mechanism has yet been obtained. In
the brain, free fatty acid and malonyl-CoA regulate feeding
behavior (46). Additionally, targeting the deletion of CPT-1c,
specifically expressed in the brain, results in obesity in mice
fed a high-fat diet (45). These reports suggest that fat metabolism is integrated into the system that monitors energy utilization and energy storage, and thus plays an important role in
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Fig. 10. Effect of 2-DG on licking behavior. A: cumulative licking counts for
corn oil and sucrose solution in mice administered 2-DG (200 mg/kg). Values
are means ⫾ SE. n ⫽ 12. B: initial licking rate. Values are means ⫾ SE. n ⫽ 12.
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MERCAPTOACETATE ATTENUATES ACCEPTANCE OF FAT
the regulation of food intake and energy homeostasis. Through
such a system, animals may evaluate fat as a food. Further
studies are needed to elucidate the mechanism by which fat is
evaluated as an energy source in the oral cavity.
Perspectives and Significance
ACKNOWLEDGMENTS
The authors thank Miyuki Satoh and Ayumi Yamada for technical assistance.
GRANTS
This study was supported by the Program for the Promotion of Basic
Research Activities for Innovative Bioscience.
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