RUNNING HEAD: Taste Modulation 1
Taste Modulation by Linoleic Acid in Rats.
Sarah Elizabeth Cheek
Steven Dwight Robinson
Dylan Burr Scott
Submitted as partial requirement
of the psychology major at Wofford College
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Taste Modulation by Linoleic Acid in Rats - Paper | PSY 451, Papers of Psychology
Material Type: Paper; Professor: Pittman; Class: Senior Thesis I; Subject: Psychology; University: Wofford College; Term: Fall 1998;
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Taste Modulation by Linoleic Acid in Rats.
Sarah Elizabeth Cheek Steven Dwight Robinson Dylan Burr Scott
Submitted as partial requirement of the psychology major at Wofford College
Abstract
Fat detection is an important part of taste preference, especially if the fat produces a change in the consumption of food. The disturbing rise in overweight individuals has prompted researchers to understand the underlying motivation behind overeating. Sensory mechanisms have been linked to over consumption and taste has been demonstrated to play a key role in food preference. Four general tastant qualities are recognized by the taste system. Studies suggest that free fatty acids contained in fat may affect taste receptor cells. Gilbertson (1998) found that free fatty acids modulate K+ channels and increase cell depolarization. This experiment defined the ability of linoleic acid to modulate tastant intake. Linoleic acid was added to solutions representative of the four tastant groups (sweet, sour, bitter, and salty) to determine if the presence of a free fatty acid can alter the licking performance of rats. Results from the experiment show that linoleic acid does alter the licking performance in rats. The licking performance was increased in sucrose, citric acid, and quanine, while licking was decreased in sodium chloride. With these results, human applications can be derived perhaps ultimately as a combatant of the effects of fat intake on obesity.
starch. This may be a factor as to why people tend not to consume many fruits since it is known that humans are more prone to enjoy sweet tastes over bitter. Sensory mechanisms have been linked to over consumption and taste has been demonstrated to play a key role in food preference. Therefore, taste transduction and the gustatory system directly impact food intake and preference. The transduction process occurs when there is a stimulus-induced excitation of taste receptor cells. Transduction initially occurs when tastant molecules interact with the taste receptors in the oral cavity, primarily with the cells of the apical membranes (Gilbertson, 1999). Most taste stimuli interact primarily with ion channels or receptors in the apical membrane of the taste cells but certain ionic stimuli may saturate in between the tight junctions of the taste cells and activate a paracellular pathway (Gilbertson, 1999). These taste stimuli / receptor cell interactions produce a depolarization of the taste cell through the activation of voltage- dependent Na+^ and K+^ channels that results in neurotransmitter release on afferent sensory neurons. These afferent gustatory neural signals encode stimulus quality and intensity (Gilbertson, 1999). It is known that the taste system can recognize at least four groups of tastants; salty, sweet, bitter, and sour. Salty tastes are detected when Na+^ ions pass into the taste cells through apical ionotropic channels causing a depolarization of the cell. Acidic tastes are detected when H+^ (protons) inhibit apical K+^ channels, reducing the outflow of K+^ and thereby depolarizing the taste receptor cell. Some sweet compounds and amino acids may bind to and activate metabotropic receptors which, coupled with ion channels, cause a depolarization of the taste cells. Bitter compounds may also act through metabotropic receptors or by directly invading the plasma membrane of the taste cell where they may
cause release of Ca2+^ from the endoplsmic reticulum, activate G proteins, or have other effects leading to an increase in cell activity such as activating transcription factors (Gilbertson, 1999). Fat is a strong source of nutrients and was believed to contain only textural cues (Gilbertson, 1998). However, recent studies suggest that free fatty acids contained in fat may affect taste receptor cells. Gilbertson found that free fatty acids modulate K+^ channels which increase cell depolarization (Gilbertson, 1998). These findings demonstrate that this would increase the potency of a tastant. A study by Nassar, et al. (2001) deduced that the affinity in humans to detect food-grade conjugated linoleic acid is associated with the ability to taste PROP. Results conclude that PROP tasters are in fact better able to detect the presence of free fatty acids when added to a high-fat food, namely vanilla ice cream, than PROP non-tasters (Nassar, 2001). Because PROP tasters have a greater number of and more densely packed taste buds, they should be able detect the existence of free fatty acids at lower detection thresholds than non-tasters. Perhaps this genetic variation in combination with transduction mechanisms is the foundation of taste preferences. Several factors such as taste, nutritional value, and post-ingestive activity influence the choice of food preferences. The orosensory and metabolic effects of fat suggest ample motivation for the ingestion of oils. Research has demonstrated that rats can detect and discriminate oils based on their nutritive value prior to ingestion (Graciela, & Sclafani, 1990). Taste preference for corn oil over mineral oil has been specifically investigated to provide evidence for the assumption that oils with the most nutritive value would be the most preferred. Long chain fatty acids are also believed to be involved in pre-ingestional discrimination (Tsuruta at el., 1999). Vegetable oils have been
PROCEDURES AND METHODOLOGY
Subjects 12 male Sprague-Dawley rats CrL;CD (SD) BR were used in the experiment. All subjects were at least nine days old and were acquired from the Charles River Breeding Laboratories. During the course of the experiment, the rats were individually housed in transparent plastic cages in a temperature-controlled colony room on a 12-12 hour light- dark cycle. All rats had free access to Harlan Teklab 2014 rodent chow and deionized- distilled water ad libitum unless otherwise specified in the methodology. Equipment A custom gustatory apparatus, the MS-160 model Davis Rig, was used to conduct the experiment. The Davis Rig consists of a chamber, to house subjects during testing and a taste stimulus delivery system, which systematically allowed access to a maximum of 16 individual bottles. The taste stimulus delivery system consisted of a motorized shutter that opened and closed at specific intervals, limiting each rat’s exposure to individual solutions. Software on the computer program monitored the duration of each presentation and recorded the exact timings of licks. This apparatus calculated the latency until the first lick, total number of licks during each presentation, and the average interval between licks. At the beginning of each testing day, the number of presentations and the order and duration of each presentation were programmed into this software package before the first subject was run. Several measures were taken to minimize the influence of non-gustatory stimuli on the data collection. Air circulation was maintained in the Davis Rig by a fan, which decreased taste stimuli odors and largely prevented
olfactory stimuli from interfering with taste preference measurements. A white noise generator minimized each subject’s exposure to auditory stimuli that may potentially interfere with licking performance. The temperature was maintained between 68° and 73°F in the experimental chamber during testing. At the end of each training day, the shutter, motor, and Davis Rig testing chamber were disassembled and all parts of the apparatus and sipper tubes were thoroughly cleaned with soap and warm water. Chemical Stimuli Four behavioral tests examined the intake patterns of a prototypical taste stimulus from each tastant quality. Intake patterns to the tastants alone and the tastants mixed with 88 μM linoleic acid were measured. Tastant solutions were prepared each day prior to testing. Each tastant was dissolved in deionized water containing 5 mM ethanol at room temperature. The addition of ethanol increased the solubility of the linoleic acid. Sucrose is representative of the sweet tastant quality and naturally reinforces licking behavior. The sucrose concentrations tested were 15, 31, 62, 125, and 250 mM. NaCl is representative of the salt tastant quality and is perceived by rats to be moderately aversive. Rats lick NaCl solutions less than water and the aversive quality increases with higher concentrations. The NaCl concentrations used were 31, 62, 125, 250, 500, and 1000 mM. Citric acid is representative of the sour tastant quality and is perceived by rats to be aversive. Similar to NaCl, rats lick citric acid less than water and the aversive quality increases with higher concentrations. The citric acid concentrations used were 1.5, 3, 7, 15, 30, and 60 mM. Quinine (QHCl) is representative of the bitter tastant quality and is perceived by rats to be extremely aversive. Even when the rats are water
Four experimental days were designated “sucrose training days,” during which sucrose solutions in 5 mM ethanol were presented until an S-curve was established and maintained for sucrose solution ingestion. These sessions served to establish a baseline of licking behavior that was compared to the number of licks for the solutions with the addition of linoleic acid. It should be noted that in sucrose testing, 125 and 250 mM were designated to three sipper bottles for a total of 12 presentations for each training session. Following this training period, four days of experimental testing sessions were administered, during which linoleic acid was added to all of the tastant solutions. During administration of aversive taste stimuli (NaCl, citric acid, and QHCl), all subjects were deprived of water for 23.75 hours prior to the beginning of each test session. When the daily testing session was concluded, all subjects were given access to water for 15 minutes. The second stimulus examined was NaCl. Six concentrations of this tastant were used. Two sipper tubes were designated to each of the six NaCl concentrations creating 12 tubes of solutions. Seven days total were devoted to this taste stimulus. The first three days enabled a baseline of licking behavior to be stabilized and established. Linoleic acid was added to the solutions (including the four tubes that had only distilled water) the last four days. All subjects were water deprived during this time frame to encourage licking behavior in the presence of a stimulus that is aversive under normal conditions. Citric acid was the third stimulus tested. Six concentrations were used and two sipper tubes were designated to each citric acid concentration. Four additional tubes contained pure distilled water, creating 16 presentations. Five days total were devoted to
this taste stimulus. The first three days were used to establish a baseline of the average number of licks at each concentration in the absence of linoleic acid. Linoleic acid was added to all solutions the last two days. Citric acid is normally regarded as aversive to the Sprague-Dawley strain of rats. Therefore, water deprivation was necessary during this experimental phase as well. Quinine (QHCl) was tested last. An identical presentation procedure was used for this taste stimulus as was observed with NaCl and citric acid. Four days total were devoted to QHCl, the first two days in the absence of linoleic acid and the last two days in the presence of 88 μM linoleic acid. Water deprivation was also used during this four- day testing period. Statistical Analysis A one-way repeated-measures ANOVA was used to determine significant main effects or interactions (p< 0.01) between the independent variables (stimulus concentration and presence of linoleic acid). A Least Significant Difference (LSD) post hoc test was used to determine the source of significant main effects. Data for NaCl, citric acid, and QHCl was standardized by calculating a tastant- water lick ratio for each of these tastant qualities. This ratio controlled for differences in lick rate and the level of motivation based on the water deprivation. A ratio of 1. indicated that the subject licked an equal amount of times to the taste stimulus and water. Ratios below 1.0 indicated the presence of aversive tastant qualities as the subject decreased licking behavior relative to water. Conversely, ratios above 1.0 would show reinforcing tastant qualities in comparison to water.
stimulus except 1000 mM NaCl. This demonstrated that linoleic acid increased the aversive quality of the salt stimulus. The decrease in licking was significantly different at 250 and 500 μM concentration. Similar to Fig. 2, Fig. 3 shows an inverted S-shaped curve. However, standard deviation bars show considerable overlap in concentrations with and without linoleic acid. Figure 4 shows the response curve for citric acid. Significant effects were seen at the 7 and 30 mM concentrations. These results contrast the theory that linoleic acid reduces ingestion of an already aversive stimulus. The unexpected increase in licking behavior for citric acid may be specific to sour tastes and related to their specific transduction mechanism.
Fig 3. The mean (ascending concentrations of QHCl alone and with 88 μM± S.E.M) ) tastant / water lick ratio of linoleic acid in rats. Stars represent significant differences (p 0.01) <
Fig 4. The mean (ascending concentrations of QHCl alone and with 88 μM± S.E.M) tastant / water lick ratio of linoleic acid in rats. Stars represent significant differences (p 0.01) <
Discussion The results clearly depict that the addition of a very small quantity of linoleic
acid has the potential to modulate taste responses to a variety of taste stimuli. The presence of linoleic acid increased the licking response to a naturally reinforcing tastant, increasing concentrations of sucrose, and conversely decreased the licking response to naturally aversive tastants, namely QHCl, NaCl, and citric acid. The stimulus presentations were only 20 seconds in duration, thus the influence of any post-ingestive signals were minimized; therefore, the changes in licking responses must be modulated solely by taste-mediated behaviors. It was concluded that linoleic acid increased the intensity of the tastant making rewarding stimuli more rewarding and aversive taste stimuli more aversive. Our behavioral data support the neurophysiological hypothesis that linoleic acid indirectly increases the potency of tastants. Sucrose is highly palatable and rats increased licking at higher concentrations of sucrose. When linoleic acid was added to the sucrose solution, the rats increased the licking response, thus responding to lower concentrations of sucrose plus linoleic acid in a similar manner as higher concentrations of sucrose alone. The increase of sucrose potency may be attributed to an observed decrease in basolateral K+^ efflux when linoleic acid is present during the transduction of sucrose (Gilbertson, 1999). Studies suggest that rats recognize fat in the oral cavity or other regions of the olfactory system, and that unsaturated fatty acids may be a potential taste stimulus (Smith, 1991). In isolated taste receptor cells, the basolateral delayed–rectifying K+^ channels were inhibited by the presence of several unsaturated fatty acids including linoleic acid (Gilbertson, 1999). The inhibition of the K+^ efflux from taste receptor cells serves to prolong the duration of depolarization during stimulus transduction, thus
No significant differences were observed between equivalent QHCl concentrations alone or with linoleic acid. Contrary to our hypothesis, data for QHCl does not give any support to the idea that linoleic acid strengthens the perception of taste stimuli. This is perhaps because quinine is already an extremely aversive stimuli as demonstrated by the low tastant-water lick ratio even at the lowest concentration of quinine (1.5 mM quinine = 0.05-0.1 ratio). Perhaps future investigations utilizing a less aversive bitter tastant might yield changes in behavior that reflect increases in bitter taste potency. Results from the experiment show that the addition of linoleic acid alters the licking performance of rats. More studies should be conducted to measure neural activity in specific taste receptor cells to confirm that the presence of linoleic acid does increase depolarization of the taste receptor cells in an intact gustatory system. Additional behavioral studies are necessary to further characterize the effectiveness of linoleic acid in modulating taste responses and food intake patterns. Studies with linoleic acid in rats should be duplicated in human subjects to determine if the results can be applied to humans. These future studies should be designed to explore the relationship between the effects of fat on taste in a rodent model and human model. If similar effects are demonstrated in a human model then the research on fat taste in the rodent model will provide an important contribution to the understanding of taste modulation by fat in relation to high fat diets. By understanding the transduction of fat taste, an application for the treatment of obesity can perhaps be derived. For example, our research shows that the level of intake of a particular solution is altered by the addition of small amounts of linoleic acid (
μM). Essentially, the potency of the tastant is increased. By applying that in the human model, it would be logical to reason that adding linolic acid to a food that is healthy, but of low palatability, would tend to increase its palatability, thus creating a healthier diet. This application must be considered further since the addition of the chemical (linoleic acid) is notably healthier than ingesting of whole fats. There are potential problems with the addition of linoleic acid to food. For example, linoleic acid must be kept cold because it oxidizes when exposed at room temperature for long durations. Food that has the addition of linoleic acid and is room temperature or warmer and not refrigerated runs the risk of becoming oxidized and changing the chemical structure of linoleic acid. Similarly, linoleic acid is not very soluble, which is why the addition of ethanol was needed. The amount of ethanol used can impact the solubility and effectiveness of linoleic acid in food. For example, the amount of linoleic acid and ethanol would vary depending on the size of the subject and has the potential to significantly alter the effectiveness of the solution. Nevertheless, the current results suggest the potential for development of healthier fat substitutes using free fatty acids such as linoleic acid as a taste enhancer.