We investigated the effect of β-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.
- licking behavior
fat is an attractive food, 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).
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 β-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 β-oxidation.
MATERIALS AND METHODS
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 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 = 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.
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 13C-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 13C-labeled 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 13C-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 μmol/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 (Sigma-Aldrich, 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 μmol/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.
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
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.
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.
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 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 μmol/kg or of 600 μmol/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 dose-dependently 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.
Effects of mercaptoacetate and 2-deoxyglucose on food intake.
The intraperitoneal mercaptoacetate administration (600 μmol/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 saline-administered 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 mercaptoacetate-administered 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 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 μmol/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 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).
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.
Mercaptoacetate is widely used to inhibit the β-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 β-endorphin precursor, proopiomelanocortin mRNA, in the hypothalamus. β-Endorphin is known to 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 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 β-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 β-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 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
We demonstrated here that the oral acceptance of fat or affinity for fat is closely related to fat metabolism. The present findings suggest that there is a novel pathway of fat recognition clearly differing from that of fundamental taste in the oral cavity. Although fat is a good source of energy, obesity due to the excessive intake of fat is a growing health problem. However, because of the high palatability of high-caloric fatty foods, humans are often unwilling to decrease their fat intake. Thus, an elucidation of the mechanism of oral acceptance of fat is expected to lead not only to an understanding of its physiological role but also to the prevention of excessive fat intake.
This study was supported by the Program for the Promotion of Basic Research Activities for Innovative Bioscience.
The authors thank Miyuki Satoh and Ayumi Yamada for technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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