Taste Sensitivity to Linoleic Acid 1 Differences in Taste Sensitivity to Linoleic Acid between Male and Female Rats

Obesity is an increasingly serious health problem in the United States, as it is responsible for serious health problems and death. Obesity is linked to ingestion of dietary fats, therefore the identification of a component of dietary fats that may increase ingestion by orosensory stimulation represents a critically important step towards controlling fat intake. Fat is composed of a combination of several different free fatty acids, which have been shown to be detectable by rats using conditioned taste aversion methodology. Linoleic acid appears to be more detectable than oleic acid, both of which have been shown to increase the licking to sweet solutions, presumably by increasing the perceived sweetness of the solutions. The goal of our research is to discover if varied levels of linoleic acid increases the palatability of sucrose, a desirable tastant, in a dose-dependant manner and whether there is a difference in sensitivity for male and female rats. Neither male nor females showed increased ingestion as the levels of linoleic acid increased, although both males and females did show an increase in licks to increasing concentrations of sucrose, as was expected. No differences between sexes were found for linoleic acid, but a sex difference was found for sucrose concentrations. Microstructure analysis showed that there are increases in the number of bursts with increasing concentrations of linoleic acid, and that the number of licks are regulated by the palatability of the solution rather than satiety. Although the experiment did not reveal a sex difference for taste sensitivity to linoleic acid paired with sucrose, it did show that females have an increased sensitivity to linoleic acid alone, which can be explored through future research. Taste Sensitivity to Linoleic Acid 3 Introduction Globally, obesity has become an increasing health problem. The World Health Organization estimates that nearly 400 million adults are obese. This chronic disorder has serious health consequences, including cardiovascular disease, diabetes, heart disease, hypertension, osteoarthritis, and some cancers (World Health Organization, 2000). Increased consumption of fat-rich foods and decreased amounts of sufficient exercise have attributed to the increased prevalence of obesity (Drewnowski, 1997). Likely causes for the elevated intake of fat may be an increase in the palatability of fatty foods, a decreased satiation in response to fats, or a combination of these two factors (Greenberg and Smith, 1996). Current research using the rat model examines the palatability of fatty foods. The rodent model has provided evidence for the detection and preference of dietary fat using corn oil as a stimulus (Greenberg and Smith, 1996). When rats were presented with mineral oil and corn oil, they showed a preference for corn oil, which is attributed to taste detection because all other sensory properties (smell, texture, and appearance) were the same for each stimuli (Greenberg and Smith, 1996). Smith et al. (2000) found that a conditioned taste aversion (CTA) to a sucrose-corn oil mixture generalized to linoleic acid. Linoleic acid (52%) and oleic acid (31%) make up a large portion of the free fatty acids (FFAs) in corn oil (Gunstone, 1999). Linoleic acid may be chemical in corn oil which can be detected through orosensory cues by the rat. The perception of FFA taste has been attributed to the role of lingual lipase in the production of FFAs from lipids. Lingual lipase can produce approximately 53 mM of oleic acid from triacylglyceride triolein after only 1 s of exposure to the lipids (Kawai and Fushiki, 2003). Fatty acid transporters (FAT) proteins bind to the FFAs, allowing interaction with the taste receptor cells (TRCs) (Fukuwatari et al. 1997). Work by Gilbertson and associates (1997) Taste Sensitivity to Linoleic Acid 4 showed evidence that free fatty acids inhibit the delayed rectifying K+ (DRK) channels in rat taste receptor cells. This activity could represent the taste cue for dietary fat. (Took out cite) Previously, conditioned taste aversions were utilized to examine the detection of free fatty acids in rats. McCormack and associates (2006) found that rats avoided both linoleic and oleic acids at concentrations greater than or equal to 66 μM and failed to avoid both 44 μM linoleic and oleic acid using 2-bottle preference tests. Therefore the behavioral detection threshold for linoleic and oleic acid is in the micromolar concentration range that is only slightly higher than the concentrations used in the Gilbertson et al. (1997) study. Previous work conducted in our laboratory has demonstrated that the ability to detect linoleic acid is eliminated when the afferent neural pathway, the chorda tympani nerve (CTdeleted N), is compromised prior to the CTA using the 2-bottle preference test paradigm. The rats that received chorda tympani nerve transection (CTX) showed no conditioned avoidance to 88 μM linoleic acid, whereas those with an intact CTN continued to avoid the LA. Other studies using short duration stimuli have shown that rats could detect and avoid concentrations of linoleic acid between 5 and 20 μM and oleic acid between 20 and 50 μM. Through CTA and CTX manipulations, the detection thresholds and neural pathway for FFAs, linoleic and oleic acids, are becoming established. Pittman and associates (2006a) found that with the addition of 88 μM linoleic acid or oleic acid to sucrose and glucose there was increased licking behavior across concentrations of those solutions. When 88 μM LA or OA was added to NaCl, citric acid, and quinine, the licking response was decreased. These results suggest that with innately appetitive taste stimuli, glucose and sucrose, the addition of FFAs enhance those positive effects, while the opposite effect occurs for aversive tastes such as, bitter, salt, and sour. When LA and OA were mixed in concentrations, Taste Sensitivity to Linoleic Acid 5 proportional to those found in corn oil, there was a similar effect on licking behavior as with the addition of either FFA individually. (Moved from beginning of this paragraph) Similarly in human studies, it has been suggested that fat causes heightened intensity ratings of other tastes. Kanarek, Ryu, and Przypek (1995) suggested that the interaction between fat and salt content might increase the hedonic value and intensity ratings by women. With increased fat content, saltiness ratings also increased. Salbe et al. (2004) found that with increasing fat content in cream solutions there was an increased creaminess rating by human participants. (Deleted paragraph starting with “Research on taste thresholds in rats....” Other research conducted by Pittman and associates (2006b) examined the differences between male and female rats in the detection of free fatty acids following a conditioned taste aversion. Both male and female rats conditioned with linoleic acid showed an avoidance at 20, 50, 75, and 100 μM LA, but the amount of avoidance differed. There was a significant difference between males and females at the 50 μM concentration of LA. In order to explore the sensitivity difference more completely, the methodology was modified to more natural feeding environment, no conditioned taste aversion was administered and the rats were water replete in the current study. The current experiment examines how linoleic acid can influence the consumption of sucrose, comparing between males and females. It is hypothesized that with addition of LA to sucrose concentrations, lick rates would increase as a function of linoleic acid. Differential sensitivity to linoleic acid between the sexes was also examined. Taste Sensitivity to Linoleic Acid 6 Methods Subjects Subjects were twelve male and twelve female Sprague-Dawley rats (greater than 90 days old, obtained from Charles River Laboratories, Wilmington, MA) housed individually in transparent plastic cages in a temperature-controlled colony room on a 12-12 h light-dark cycle with lights on at 7:00 h. Rats had free access to Harland Tekland 8604 rodent chow. Rats were given deionized, distilled water ad libitum during all testing days, and were restricted of water during the two days of training. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Wofford College. Apparatus Testing was conducted in the MS-160 gustatory apparatus (DiLog Instruments, Tallahassee, FL). The “Davis Rig” allows the controlled presentation of 16 chemical stimuli and records the licking behavior of the rat at 1-ms resolution. Taste stimuli were presented for controlled durations, wait periods, and interstimulus intervals using the sliding stimulus rack and the controlled lever, opening and closing the access port. The Davis Rig is located in an acoustic isolation chamber with fans that maintain a constant flow of air, in order to minimize olfactory cues. The chamber is lighted with an 8-watt, and rat behavior is monitored using a real-time internet camera. Phase 1 Two days prior to testing day, the rats were trained in the Davis M-s 160 gustatory apparatus, a device that measures licking patterns to various taste stimuli. During testing, the rats were given access to four bottles of taste stimuli in fifteen second presentations. There were Taste Sensitivity to Linoleic Acid 7 thirty-two testing trials comprised of eight blocks of stimuli of four stimulus groups. As demonstrated in Table 1, both male and female rats were grouped in a counter-balanced design of stimulus presentations. Table 1. Counter-balance design of stimulus presentations. Day 1 Day 2 Day 3 Day 4 Sucrose Group 1 Group 2 Group 3 Group 4 Sucrose + 200 uM LA Group 2 Group 3 Group 4 Group 1 Sucrose + 400 uM LA Group 3 Group 4 Group 1 Group 2 Sucrose + 800 uM LA Group 4 Group 1 Group 2 Group 3 Analysis The number of licks (consumption) and latency until the first lick was recorded for each trial. The dose dependent effects of adding linoleic acid at various sucrose concentrations during short fifteen second trials were calculated for the male and female Sprague-Dawley rats using a lick ratio that normalized taste stimuli responses to each animal’s average daily response to water trials, such that a lick ratio of 1.0 is identical to water licking and zero is no response. A mixed factorial ANOVA was used to examine the between-subject effects of sex and with-in subject effects of sucrose concentrations and linoleic acid concentrations. Phase 2 Twelve male Sprague-Dawley rats were repleted of water prior to testing. The rats were tested across two days in the Davis M-s 160 gustatory apparatus. On each day of testing, the rats were given access to the sixteen taste stimuli in one 90 second presentation.