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Transient Overconsumption of Novel Foods by Deafferentated Rats
Physiology & Behavior, Vol. 65, Nos. 4/5, pp. 793–800, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99 $–see front matter
PII S0031-9384(98)00237-6
Transient Overconsumption of Novel Foods by Deafferentated Rats: Effects of Novel Diet Composition LISA A. KELLY, MARK CHAVEZ AND HANS-RUDOLF BERTHOUD1 Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Baton Rouge, LA, 70808 Received 11 November 1997; Accepted 6 August 1998 KELLY, L. A., M. CHAVEZ AND H.-R. BERTHOUD. Transient overconsumption of novel foods by deafferentated rats: Effects of novel diet consumption. PHYSIOL BEHAV 65(4/5) 793–800, 1999.—We recently demonstrated that capsaicin-treated rats consume more of an unfamiliar high-fat diet than vehicle-treated controls, but only on initial exposure (Chavez et al, 1997). We hypothesized that negative feedback signals carried by capsaicin-sensitive visceral afferents are critical for the regulation of intake of novel foods, but redundant pathways take over during subsequent exposures. To examine the role of nutrient content of the novel diet, rats were systemically treated with capsaicin (n 5 15) or vehicle (n 5 10), and exposed to 1) a fat/olestra diet that was isocaloric with chow; 2) a readily accepted fat-free cake; and 3) pure corn oil. Each 3-h feeding trial was preceded by 24-h food deprivation. Treated rats did not overconsume familiar chow, but did consume 50% more than controls of both the fat/olestra diet and the corn oil on first exposure; this suggests that capsaicin eliminated visceral afferents that normally carry satiety signals. However, the effect with the fat/olestra mixture was due primarily to depressed intake by controls, unlike the pure fat diets; this apparent neophobic response was blunted in treated rats. Because treated rats failed to overconsume the fat-free cakes, the neural system damaged by capsaicin appears to be linked to energy or fat sensory mechanisms, and possibly to hedonic responsiveness. © 1999 Elsevier Science Inc. Satiety Visceral afferents Food neophobia
High-fat diets
High carbohydrate diets
size and short-term food intake (4,25,30), it has not been possible to clearly demonstrate such a feeding effect with surgical or chemical ablation of vagal or visceral afferents (7,20,22). This failure is likely due to the extended recovery period necessary between ablation and testing, with the resulting development of compensatory satiety mechanisms; for this reason, a nontraumatic acute procedure would be ideal. We have further hypothesized that the salience of visceral afferent satiety signals may be minimal after a food has become familiar, because control of intake shifts to gustatory cues through learned associations. In support of this view, we have recently demonstrated that capsaicin-treated rats consume more of an unfamiliar high-fat diet than do vehicle-treated controls, and that this overconsumption occurs only on initial but not subsequent exposures (8). These findings are consistent with the idea that during ingestion of novel, but not familiar foods, negative feedback signals are carried by capsaicin-sensitive
THE consumption of food elicits a cascade of sensory signals as it is ingested, digested, absorbed, and metabolized. There is evidence for a number of vagal and spinal mechano- and chemosensors along the gastrointestinal tract and the portal hepatic axis; in addition, nutrients and their metabolites elicit the secretion of hormones and local messengers. It is thought that positive or reinforcing feedback signals are used by the brain to augment or maintain current ingestion (11,36,43), and negative feedback signals tend to suppress intake and accelerate the satiation process (14,16–18,33,39,41). In addition, there is increasing evidence that these various classes of sensory signals are used to form memorial representations and associations that guide future ingestion (3,10,12,15,23,27,42). Experimental support for these general concepts is still incomplete. For example, while it has been shown that blockade of vagal satiety pathways in the periphery by administration of selective CCK-A receptor antagonists can increase meal
1 To
Deprivation-induced feeding
whom requests for reprints should be addressed. E-mail: [email protected]
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visceral afferents critical for initial ingestion, but that redundant, capsaicin-resistant pathways take over this task during subsequent exposures. The novel test foods in our recent study were palatable, high-energy, high-fat foods, including a pure-fat diet and a complete liquid diet (Ensure), chosen specifically to make the new diet as different as possible from the maintenance diet. Thus, the aim of the present study was to determine which characteristics of the novel test food are necessary and sufficient to produce the overconsumption response in our experimental paradigm. For example, it may be essential that the test food have a higher energy density than the maintenance diet, or a different flavor, or mouth feel. South and Ritter (40) report that a highly palatable food (sucrose solution) must be used to observe intake differences between control, and either AP/NTS lesioned or fourth ventricular capsaicin-treated rats, while Curtis and Striker (9) demonstrated that rats treated with peripheral capsaicin also overconsumed a sucrose solution. Similarly, close examination of findings by Castonguay and Bellinger (7) reveal that capsaicin-treated rats initially overconsumed a novel sweetened milk diet, in comparison to control rats; in contrast, total intake and meal size of familiar chow was similar for the two groups. The role of macronutrient content in the induction of this behavioral effect is also unknown, as the fat content of both test diets in the initial study was much higher than that of the low fat chow maintenance diet. To address these issues of diet characteristics in the present study, vehicle- and capsaicin-treated rats maintained on chow were exposed to various novel foods after 24-h food deprivations. For Experiment 1, a pure fat/olestra mixture of the same caloric density as chow was used as the novel diet. In Experiment 2, the same rats were exposed to fat-free, highcarbohydrate cakes as the novel test diet to determine if the differential feeding response is retained with an unfamiliar food that is composed of carbohydrate rather than fat. MATERIALS AND METHODS
Animals Twenty-five male Sprague–Dawley rats (Harlan Industries, Indianapolis, IN) were used, weighing 180–250 g at the time of capsaicin- or vehicle-treatment. The animals were housed individually in hanging wire-mesh cages under standard laboratory conditions (12:12-h lighting schedule, lights on at 0700 h, 22638C), and 5001 Purina lab chow and tap water available ad lib except as noted prior to tests. All testing was conducted in the light phase between 1100 and 1400 h. Capsaicin Treatment Rats were treated consecutively with increasing doses of capsaicin. On each of 3 days, rats were injected under inhalation anesthesia (isoflurane) with either vehicle as a control, or a dose of capsaicin (12.5, 30, and 75 mg/kg, i.p., Sigma Chemicals, 98% grade). Capsaicin was dissolved freshly in a mixture of Tween 80 (10%), ethanol (10%), and sterile saline (80%) at the specific concentration. Following the first injection all rats exhibited respiratory arrest of between 1 and 5 min. In most cases, assistance by manually massaging the chest was sufficient to resume spontaneous breathing. In the remaining cases artificial respiration with oxygen was applied. One rat died during the first injection. During subsequent injections with the higher doses artificial respiration was rarely necessary.
Eye-Wipe Test Ten days following capsaicin treatment, one drop of 1% NH4OH was applied to the left eye with a Pasteur pipette and the number of eye wipes in 30 s and the latency to the first wipe were recorded. All capsaicin-treated rats fulfilled the criterion of less than three wipes and a latency of greater than 5 s to the first wipe. All vehicle control animals wiped vigorously, with a latency of less than 1 s and greater than 15 wipes/30 s. Short-Term Feeding Tests Begun 6 weeks after capsaicin treatment, Experiment 1 consisted of a series of five short-term feeding tests, performed at intervals of 6 days, each preceded by 24 h of food deprivation. The first test was conducted with the familiar pelleted chow diet, and the remaining four trials with a mixture of vegetable shortening (Crisco) and Olestra (a sucrose polyester, Proctor & Gamble) at a ratio of 1:1 by weight, resulting in a solid greasy diet mimicking the mouth feel of pure shortening, and isocaloric with the maintenance chow diet (3.4 kcal/g). On the day before the first test with the new diet, a small amount (,0.5 g) of the new diet was offered in an attempt to reduce any potential neophobic response to the food. The test food was given at 1100 h for a 3-h period, then chow was returned to the animals 5 h later at dark onset and overnight intake measured. During each trial, intake was measured at 30, 60, 120, and 180 min from the first bout of ingestion by weighing the food jar and spillage. Olestra has been known to cause soft stools, and because we noted that numerous rats of both groups showed soft feces after the first exposure to fat/olestra, the 24-h total wet weight of feces was measured for every trial afterward. Soft stool is a possible indicator of gastrointestinal comfort produced by Olestra. Individual cardboard spill pads were placed beneath each wire cage to collect the feces, and weighed immediately at the conclusion of the 24-h period. Soft feces were defined as any stool that did not appear as a firm pellet on visual inspection. Experiment 2 consisted of a second series of similar shortterm feeding trials using the same animals after a period of 4 weeks of ad lib access to chow. Following an initial test with chow, two tests with a commercially available fat-free dessert cake (Entenmann’s, 91.4% carbohydrate, 8.6% protein, 2.5 kcal/g) was used as the novel diet, because it has been reported that rats readily consume this food (35). Because no overconsumption response in capsaicin-treated rats was found with this novel food, a final test with pure corn oil (8.5 kcal/ mL) as the unfamiliar food was carried out. A comparison of energy density and macronutrient composition of the various diets is presented in Table 1. CCK Intake-Suppression Test After completion of all feeding tests, approximately 23 weeks following capsaicin treatment, animals were tested for the known food intake-suppression effects of exogenous CCK. In overnight food-deprived capsaicin- and vehicletreated rats, CCK (6 mg/kg, i.p.) or saline as a control, was administered 30 min before access to chow, and 1-h intake was measured. Half the animals of each group received CCK first, the other half received saline first. Data Analysis and Statistical Procedures The cumulative 3-h food intake results for each of the trials were analyzed independently, using a mixed-model ANOVA approach with treatment and time as factors. Comparisons of
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TABLE 1 ENERGY DENSITY AND MACRONUTRIENT COMPOSITION OF VARIOUS DIETS USED Novel Diets High-Fat
Energy density (kcal/g) % energy from fat % energy from COH % energy from protein
Fat-Free
Maintenance Chow Diet
Isocaloric to Chow (Olestra)
Corn Oil
Cakes
3.4 12 62 26
3.4 100 0 0
8.5 100 0 0
2.3 0 91 9
intake between chow trials and novel diet trials were also analyzed by a mixed-model ANOVA, with treatment as a fixed effect, time as a random effect, and trial as a repeated effect. When a significant effect of treatment or an interaction was found, Bonferroni t-tests were used in the post hoc pairwise analyses to isolate the effect. Results of the CCK test were analyzed using paired t-tests. RESULTS
Experiment 1 Capsaicin-treated rats weighed approximately 20 g less than the vehicle-controls 2 weeks following treatment. This initial small but significant weight difference was then maintained throughout the duration of the study, as both groups gained weight at a similar rate. Such a lasting difference in body weight was not observed in our earlier report and other experimental cohorts. During the first 3-h test with the familiar chow diet, capsaicin- and vehicle-treated rats consumed similar amounts (Fig. 1). In contrast, on the first trial with the new fat/olestra diet, capsaicin-treated rats consumed significantly more than controls, F(1, 19) 5 8.56, p , 0.01. Follow-up Bonferroni’s tests revealed that the treated rats consumed significantly more at both 60 and 120 min (pls , 0.05). If intake is compared to that of the preceding chow trial, it can be seen that the control rats showed depressed intake of the new diet, F(1, 246) 5 45.83,
p , 0.001, while the capsaicin-treated rats consumed similar amounts of chow and fat/olestra. Because it could be argued that the lower body weight of the capsaicin-treated rats may be responsible for differences in the short-term food intake trials, we analyzed 1-h food intake of the two groups with an adjustment for body weight. Using a model that allows different variances for the two treatment groups and Satterthwaite’s approximation of degrees of freedom, the analysis yielded a significant difference in 1-h food intake due to treatment, F(16, 8) 5 9.88, p , 0.01. Therefore, the overconsumption of olestra/fat diet by capsaicin-treated rats in the first trial was not due to differences in body weight. During the second exposure to the new diet, 6 days later, the consumption levels were reversed, such that the capsaicin treated rats now consumed less than the controls, F(1, 19) 5 10.21, p , 0.01. Post hoc comparisons revealed that intakes were significantly different at 1, 2, and 3 h. When compared with the consumption during the first exposure to the new diet, it can be seen that the control rats increased their intake, while that of the treatment group decreased. Despite this lower olestra intake, the capsaicin-treated rats showed a significantly higher wet weight of fecal output (22 vs. 16 g; p , 0.05; Fig. 2), and a higher incidence of soft feces (84% in treatment group vs. 40% in control group). In contrast, measurement of 24-h feces output during the second chow trial showed no difference between the groups in either feces weight or appearance. Olestra is well known to induce soft
FIG. 1. Mean (6SEM) 24-h deprivation-induced cumulative intake of chow (left panel) and solid fat/olestra (first and third exposures) in a series of short-term feeding trials. Vehicle rats (n 5 10) are represented by the solid line, and capsaicin-treated (n 5 13) by the broken line. Maintenance chow was returned 5 h after the conclusion of the test, at dark onset; trials were separated by 6 days. *p , 0.05 (Bonferroni’s correction).
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KELLY, CHAVEZ AND BERTHOUD stools (2,26), but our results further indicate that the bowel response to the fat substitute was aggravated in capsaicintreated rats. Capsaicin-treated rats continued to significantly underconsume the olestra diet during a third, F(1, 19) 5 10.21, p , 0.01, and fourth, F(1, 19) 5 16.89, p , 0.01, trial. In these two additional trials, the pattern of intake for both groups was similar to the second trial shown in Fig.1. Experiment 2
FIG. 2. Each bar represents mean (6SEM) 24-h fecal output after a short-term feeding trial: second exposure to fat/olestra, paired bars on the left, and chow, pair on the right. Vehicle rats (n 5 10) are represented by the left-hand bar and capsaicin-treated (n 5 13) by the right-hand bar. The lower, striped portion of each bar indicates the fecal output during the 3-h test period, while the upper solid portion shows the output during the remaining 21 h. Feces were collected on individual spill pads placed beneath the wire cage housing each rat, and weighed immediately at the conclusion of each period. *p , 0.05 (Bonferroni’s correction).
As in Experiment 1, the capsaicin-treated and control rats consumed almost identical amounts of chow, with similar time courses (Fig. 3). However, in contrast to Experiment 1 with the high-fat diet, capsaicin-treated rats did not overconsume the fat-free high-carbohydrate cakes on first exposure; rather, there was a small but significant treatment effect on food intake, F(1, 16) 5 6.17, p , 0.05, indicating higher consumption by controls, but with no individual time point showing a significant difference. Compared with the chow trial, both groups of rats consumed significantly more calories of the fatfree cakes, F(1, 140) 5 14.31, p , 0.001; F(1, 140) 5 61.61, p , 0.001. The second trial with this new food showed essentially the same results, with no significant difference in intake either between the groups or between the trials. During a final trial, which exposed the rats to pure corn oil 6 days after the last trial with fat-free cakes, the capsaicintreated rats overconsumed relative to both the controls, F(1, 18) 5 15.58, p , 0.001, and their own previous chow consumption, F(1, 140) 5 155, p , 0.001. Unlike the fat/olestra
FIG. 3. Mean (6SEM) 24-h deprivation-induced cumulative intake of chow (upper left panel), fat-free cake crumbs (Entenmann’s brand), and pure corn oil in consecutive short-term feeding trials. Vehicle rats (n 5 10) are represented by the solid line, and capsaicin-treated (n 5 11) by the broken line. Maintenance chow was returned 5 h after the conclusion of the test, at dark onset; trials were separated by 6 days. *p , 0.05 (Bonferroni’s correction).
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trial, the energy intake of control rats was not depressed relative to chow levels; on the contrary, it was slightly but significantly higher, F(1, 146) 5 6.97, p , 0.01. The patterns of intake were different between the two groups, as the treated rats exhibited essentially all consumption within the first 30 min, whereas the control group continued to consume corn oil throughout the 3-h trial.
Because the effect reappeared on exposure to the corn oil 6 days later, the loss cannot be attributed to experimental history. However, it remains to be clearly demonstrated that it is the macronutrient content in the cake that caused the loss of the overconsumption effect, rather than either energy density or palatability. For example, we have shown that the largest, absolute overconsumption (with energy intake larger than that of chow) occurs with high energy diets [solid fat (8) and corn oil (present results), of about 8 kcal/g], while the medium energy diets—Ensure (8) and fat/olestra (present results)— induce only a relative overconsumption that results from depressed intake by the control rats. Because the fat-free cakes with the lowest energy density of 2.3 kcal/g failed to induce any overconsumption, one might argue that high-energy density rather than fat is needed for the novel diet to cause overconsumption in capsaicin-treated rats. However, we have recently found that capsaicin-treated rats overconsume corn oil emulsion with a low-energy density of 0.8 kcal/mL by 70% (personal observations). Therefore, the lack of overconsumption of the fat free cakes is not likely due to the low energy content of this novel diet. Furthermore, the idea that high energy content of the novel diet is an important prerequisite for absolute overconsumption to occur does not appear to be supported by the results of another recent study, where 10% sucrose (0.45 kcal/mL) was consistently overconsumed by capsaicin-treated rats (8). South and Ritter (40) also reported that a sucrose solution induced overconsumption in a model with fourth ventricular capsaicin administration. Although we have not yet tested sucrose solutions for overconsumption in our model, we have no reason to assume that it would not be effective, given that Curtis and Stricker (9) used the same capsaicin treatment and the same strain of rats. Thus, the question becomes: why did solid carbohydrate not induce overconsumption, while the sucrose solution did? As it is well recognized that hydration of carbohydrate renders it extremely palatable to rats (29), it is possible that a solid form of carbohydrate cannot induce the overconsumption response due to lesser palatability than either sucrose solutions or fat-based diets. One useful approach in dissection of the role of macronutrients in the overconsumption response would be to isolate the effects of taste or palatability from those of the postingestive consequences. For example, duodenal preloads of carbohydrate or fat could be given prior to measuring food deprivation-induced intake of a novel food to directly determine whether capsaicin-treatment differentially interferes with the generation of negative feedback from each macronutrient.
CCK-8 Feeding Suppression Test CCK-8 suppressed intake of chow in the overnight fooddeprived rats by about 50% (t 5 8.9, p , 0.05; Fig. 4) in vehicle-treated control rats, but failed to induce significant suppression in capsaicin-treated rats (t 5 1.78, p 5 0.11). DISCUSSION
Similar to the results of our previous report with a novel high-energy diet of pure vegetable shortening (8), capsaicintreated rats overconsumed, relative to vehicle-treated controls, two other novel high-fat diets on first exposure: a fat/ olestra mixture and pure corn oil. In contrast, an unfamiliar, fat-free high carbohydrate food was not overconsumed. These results could be interpreted as evidence that fat is a necessary component for a novel food to induce an overingestion response in capsaicin-treated rats. Because systemic capsaicin treatment is known to destroy a population of fine, unmyelinated visceral afferents (1,19), the phenomenon of overingestion might then best be explained by the loss of negative feedback signals from ingested fat that normally suppress further intake in the intact animal. In addition, the fact that the overconsumption is transient, as it is only observed during the first 3-h exposure to the novel diet, suggests that the animal does not exclusively rely on this capsaicin-sensitive mechanism, but uses redundant pathways to recalibrate future intake of the new diet. However, before we can adopt such a simple interpretation of the results, several issues must be addressed. Role of Diet Composition In contrast to the results with the novel fat food in Experiment 1, and our previous findings with vegetable shortening and Ensure, the capsaicin-treated rats did not overconsume the novel fat-free, high-carbohydrate cakes on first exposure.
Does Capsaicin Treatment Interfere With the Expression of Normal Food Neophobia?
FIG. 4. Effect of exogenous CCK (6 mg/kg, i.p, striped bars) or saline (solid bars) on 30-min chow intake of vehicle (n 5 10) and capsaicin-treated (n 5 13) rats. Overnight food-deprived rats were injected 30 min prior to access to food. Note that food intake suppression by CCK is completely abolished in capsaicin-treated rats. Tests were conducted 23 weeks following capsaicin treatment. *p , 0.01 (t-test).
Because the phenomenon of relative overconsumption, as reported here, is dependent on decreased intake by the control rats, our hypothesis of blunted negative feedback is insufficient to account for this. Therefore, we propose two additional mechanisms that may also contribute to the differential intake levels observed between treated and control rat. First, it appears that capsaicin treatment interferes with the normal expression of food neophobia. It is well established that most novel foods are cautiously approached by rats (6). Before any oral sampling, the smell of food can already be recognized as novel, so that the animal may not touch the food for a considerable time. Eventually, small amounts are ingested for the purpose of detecting any adverse postingestive effects. Although there is an interaction of this caution
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with the level of hunger (37), 24-h food deprivation is not sufficient to overcome it. If capsaicin treatment decreased sensitivity to smell and taste, this could explain the apparent interference with neophobic hypophagia. It has been reported that capsaicin treatment does not cause a functional loss in tasting ability (38), but the authors concurrently observed that the compound action potential recorded from the olfactory nerve in response to various odor stimuli was decreased. Given that postingestive consequences must be experienced to overcome neophobia (5,28), it is unlikely that the capsaicin-induced damage to postingestive sensory mechanisms resulted in a quicker attenuation of gustatory neophobia; rather, it is more plausible that capsaicin treatment interferes with either smell or taste during the active phase of neophobia, or with brain stem mechanisms that translate neophobia into the inhibition of food intake. In light of this, it is possible that the fat-free cake failed to induce differential consumption by the treated rats because it did not provoke a neophobic response in the control rats.
15 min, these authors concluded that it is the attenuation of early signals of gastric distension that induces overingestion by treated rats. These opposing interpretations may be reconciled with the proposal that low and high threshold subpopulations of gastric distension afferents are differentially sensitive to capsaicin, and that a population of low-threshold fibers may be most susceptible to the neurotoxin. Because the above-cited studies demonstrating capsaicin-resistant vagal fibers used only relatively large distension volumes, the results, therefore, cannot rule out the existence of capsaicin-sensitive low-threshold fibers. That relatively small volumes of gastric distension can suppress intake in intact rats has also recently been demonstrated by Davis and co-workers (13). Furthermore, in addition to the loss of a directly inhibitory effect on intake via low-threshold gastric distension afferents, more distal vagal receptors could also indirectly affect intake via slowing of gastric emptying. There is evidence that small intestinal capsaicin-sensitive afferents mediate feedback inhibition of gastric emptying (46).
Does Capsaicin Treatment Enhance the Positive or Reinforcing Aspects of Foods?
Implications for Learning and Conditioned Satiety
It has been suggested that meal size, and thus, short-term control of food intake is determined by balance between positive and negative feedback signals (39). Therefore, overingestion by capsaicin-treated rats could be the result of either decreased negative feedback, as earlier hypothesized, or else increased positive feedback. This, in turn, implies that the treatment may result in the amplification of positive or reinforcing properties of food (12,24,44). A more precise examination of the microstructure of the pattern of overconsumption, either with a variety of test diets, or with a highly palatable maintenance food, is one important step in discriminating between these mechanisms underlying the overconsumption. In addition, opioid receptor dependency of the overconsumption effect should be tested in future experiments, as it is known that opioid receptors are involved in the mediation of reinforcing or rewarding properties of foods (21,45). What Is the Neurological Substrate Damaged by Capsaicin Treatment and What Portion of This Substrate Is Responsible for Overconsumption? From a more functional point of view, the above discussion implies that capsaicin damage may have impinged on a number of neural pathways, not necessarily limited to primary afferent fibers. Anatomical studies show that systemic capsaicin treatment in adult rats leads to degeneration of a distinct population of visceral afferents, including but not limited to those of dorsal root and nodose-vagal origin (1,19,34). In contrast, another population of fine unmyelinated fibers of at least vagal origin and innervating primarily the upper gastrointestinal tract seems to remain both physically and functionally intact (1,32). Specifically, it has been shown by various methods, including anatomical tracing, c-fos expression, and electrical unit recording (1,32), that many vagal afferents sensitive to gastric distension are capsaicin resistant, so that there may be only partial loss of negative feedback signals from gastric distension receptors in capsaicin-treated rats. This also suggests that the loss of signals from duodenal nutrient sensors may be the more likely explanation for their overingestion of novel foods. This conclusion, however, is in direct contrast to the one reached by Curtis and Stricker (9); based on their findings that capsaicin-treated rats overconsumed a variety of solutions, including plain tap water, and did so within the first 5 to
One striking finding from both our current and previous studies is the consistent loss of the overconsumption effect by the second and third trials with the novel food. In Experiment 1 of the present study, this could have been due to malabsorption of the fat/olestra diet, as indicated by the soft feces, which appeared to be aggravated by capsaicin treatment. The resulting gastrointestinal malaise could have produced a mild taste aversion. Similarly, in our previous study, the capsaicintreated rats ingested so much of the vegetable shortening that they may have experienced indigestion, thus producing a mild aversion during the next trials that could have interfered with possible overconsumption. However, such arguments are harder to make for one of the experiments reported earlier (8), using the nutritionally complete liquid diet Ensure as the novel food, for which there was also no sign of overingestion during the second and third trials. Furthermore, in support of our finding, closer inspection of data shown by Castonguay and Bellinger (7) indicates that while capsaicin-treated rats indeed consumed more of a novel milk diet than controls on first exposure, intakes were almost identical during the third daily 30-min exposure. We suggest that the initial overconsumption seen with capsaicin-treated rats disappears on repeated exposure due to postingestive consequences. We further believe that these capsaicin-resistant signals are also the reason that both capsaicin-treated rats do not overconsume the familiar chow in our studies, and that Castonguay and Bellinger (7) did not detect long-term changes in meal size with the chow diet. In contrast to this transient character of the overingestive response in our experimental setting, as well as that of Castonguay and Bellinger (7), Curtis and Stricker (9) observed that their capsaicin-treated rats still significantly overconsumed sucrose during the third daily 30-min test. However, procedural differences between our study and theirs may explain the apparent discrepancy. Although our paradigm uses 24-h food deprivation from a maintenance chow diet, Curtis and Striker use a restricted feeding schedule with 10 g of chow per day, and daily access of only 30 min to the sucrose solution, after 2 days continuous access. These procedures may have created considerable differences in the levels of hunger and nutritional expectation before presentation of the test food, which interfered with the recalibration process. Thus, it is possible that it may simply take more than three 30-min ex-
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posures within this paradigm for recalibration to occur. Alternatively, it is also possible that the low-energy density of the 10% sucrose induced only weak postingestive consequences, which were unable to modify subsequent intake in capsaicintreated animals. It seems clear that capsaicin treatment permanently destroys a substantial number of several types of visceral afferents, as the anatomical evidence is supported by the finding that capsaicin treatment induces a permanent loss of the effectiveness of CCK to suppress intake (31). Thus, for treated rats to exhibit such eventual control of short-term intake as we and Castonguay and Bellinger (7) report, there must exist a redundant satiety system. Furthermore, given this reasoning, the redundant pathways must not be capsaicin sensitive. In
fact, the existence of capsaicin-resistant, probably nonvagal, sensory pathways from the alimentary canal to the brain has been demonstrated by Lucas and Sclafani (23), who showed that capsaicin treatment did not interfere with the development of flavor preferences due to duodenal infusions. Thus, these pathways represent the prime candidates for functionally replacing other visceral efferents damaged by capsaicin. ACKNOWLEDGEMENTS
We would like to thank Dr. Julia Volaufova for advice and help on statistical analysis. This work was partially supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-47348 to H.-R.B., and DK-32089 to G.A.Bray).
REFERENCES 1. Berthoud, H.-R.; Patterson, L. M.; Willing, A. E.; Mueller, K.; Neuhuber, W. L.: Capsaicin-resistant vagal afferent fibers in the rat gastrointestinal tract: Anatomical identification and functional integrity. Brain Res. 746:195–206; 1997. 2. Blackburn, H.: Olestra and the FDA. N. Engl. J. Med. 334:984– 986; 1996. 3. Booth, D. A.: Conditioned satiety in the rat. J. Comp. Neurol. 81:457–471; 1972. 4. Brenner, L.; Ritter, R.C.: Peptide cholesystokinin receptor antagonist increases food intake in rats. Appetite 24:1–9; 1995. 5. Buro sˇ ová, O; Bure sˇ , J.: Post-ingestive interference with brain function prevents attenuation of neophobia in rats. Behav. Brain Res. 1:299–312; 1980. 6. Carroll, M. E.; Dinc, H. I.; Levy, C. J.; Smith, J. C.: Demonstrations of neophobia and enhanced neophobia in the albino rat. J. Comp. Physiol. Psychol. 89:457–467; 1975. 7. Castonguay, T. W.; Bellinger, L. L.: Capsaicin and its effects upon meal patterns, and glucagon and epinephrine suppression of food intake. Physiol. Behav. 40:337–342; 1987. 8. Chavez, M.; Kelly, L.; York, D. A.; Berthoud, H.-R.: Chemical lesion of visceral afferents causes transient overconsumption of unfamiliar high-fat diets in rats. Am. J. Physiol. 272:R1657– R1663; 1997. 9. Curtis, K. S.; Stricker, E. M.: Enhanced fluid intake by rats after capsaicin treatment. Am. J. Physiol. 272:R704–R709; 1997. 10. Davidson, T. L.; Altizer, A. M.; Benoit, S. C.; Walls, E. K.; Powley, T. L.: Encoding and selective activation of “metabolic memories” in the rat. Behav. Neurosci. 111:1–17; 1996. 11. Davis, J. D.; Levine, M. W.: A model for the control of ingestion. Psychol. Rev. 84:379–412; 1977. 12. Davis, J. D.; Perez, M. C.: The acquired control of ingestive behavior in the rat by flavour-associated post-ingestional stimulation. Physiol. Behav. 54:1221–1226; 1993. 13. Davis, J. D.; Smith, G. P.; Sayer, J. L.: Reduction of intake in the rat due to gastric filling. Am. J. Physiol. 272:R1599–R1605; 1997. 14. Deutsch, J. A.: The role of the stomach in eating. Am. J. Clin. Nutr. 42:1040–1043; 1985. 15. Deutsch, J. A.; Tabuena, J. A.: Learning of gastrointestinal satiety signals. Behav. Neural Biol. 45:292–299; 1986. 16. Friedman, M. I.: Control of energy intake by energy metabolism. Am. J. Clin. Nutr. (Suppl.) 62:1096S–1100S; 1995. 17. Geary, N.; Le Sauter, J.; Unkyung, N.: Glucagon acts in the liver to control spontaneous meal size in rats. Am. J. Physiol. 33:R116– R122; 1993. 18. Greenberg, D.: Intestinal satiety. In: Smith, G. P.; Gibbs, J., eds. Structure and mechanisms of satiety. London: Oxford University Press; 1996. 19. Holzer, P.: Capsaicin: Cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol. Rev. 43:143–172; 1991. 20. Kraly, F. S.; Jerome, C.; Smith, G. P.: Specific postoperative syn-
21. 22. 23. 24. 25. 26.
27. 28. 29. 30. 31. 32. 33.
34.
35. 36.
dromes after total and selective vagotomies in the rat. Appetite 7:1–17; 1986. Levine, A. S.; Weldon, D. T.; Grace, M.; Cleary, J. P.; Billington, C. J.: Naloxone blocks that portion of feeding driven by sweet taste in food-restricted animals. Am. J. Physiol. 37:R248–R252; 1995. Lorenz, D. N.: Effects of gastric filling and vagotomy on ingestion, nipple attachment and weight gain by suckling rats. Dev. Psychobiol. 16:469–483; 1983. Lucas, F.; Sclafani, A.: Capsaicin attenuates feeding suppression but not reinforcement by intestinal nutrients. Am. J. Physiol. 270:R1059–R1064; 1996. Lucas, G. A.; Timberlake, W.: Interpellet delay and meal pattern in the rat. Physiol. Behav 43:259–264; 1988. Meisner, J. A.; Smith, G. P.; Gibbs, J.; Tyrka, A.: Intravenous infusion of CCK-A receptor antagonist increases food intake in rats. Am. J. Physiol. 262:R216–R219; 1992. Mellies, M. J.; Jandacek, R. J.; Taulbee, J. D.; Tewksbury, M. B.; Lamkin, G.; Baehler, L.; King, P.; Boggs, D.; Goldman, S.; Gouge, A.; Tsang, R.; Glueck, C. J.: A double-blind, placebocontrolled study of sucrose polyester in hypercholesterolemic outpatients. Am. J. Clin. Nutr. 37:339–346; 1983. Mook, D. G.: Oral factors in appetite and satiety. Ann. NY Acad. Sci. 575:265–278; 1989. Pelchet, M. L.: What is “mere exposure”? Proceedings of the Annual Meeting of the Society for the Study of Ingestive Behavior, Baltimore, MD; 1997. Ramirez, I.: Feeding a liquid diet increases energy intake, weight gain and body fat in rats. J. Nutr. 117:2127–2134; 1987. Reidelberger, R. D.; O’Rourke, M. F.: Potent cholecystokinin antagonist L 364718 stimulates food intake in rats. Am. J. Physiol. 257:R1512–R1518; 1989. Ritter, R. C.; Ladenheim, E. E.: Capsaicin pretreatment attenuates suppression of food intake by cholecystokinin. Am. J. Physiol. 248:R501–R504; 1985. Ritter, R. C.; Ritter, S.; Ewart, W. E.; Wingate, D. L.: Capsaicin attenuates hindbrain neuron responses to circulating cholecystokinin. Am. J. Physiol. 257:R1162–R1168; 1989. Ritter, R. C.; Brenner, L.; Yox, D. P.: Participation of vagal sensory neurons in putative satiety signals from the upper gastrointestinal tract. In: Ritter, S.; Ritter, R. C.; Barnes, C. D., eds. Neuroanatomy and physiology of abdominal vagal afferents. Boca Raton, FL: CRC Press; 1992:221–248. Ritter, S.; Dinh, T. T.: Capsaicin-induced neuronal degeneration; Silver impregnation of cell bodies, axons, and terminals in the central nervous system of the adult rat. J. Comp. Neurol. 271:79– 90; 1988. Sclafani, A.; Weiss, K.; Cardieri, C.; Ackroff, K.: Feeding response of rats to no-fat and high-fat cakes. Obesity Res. 1:173– 178; 1995. Sclafani, A.: How food preferences are learned: Laboratory animal models. Proc. Nutr. Soc. 54:419–427; 1995.
800 37. Shephard, R. A.; Estall, L. B.: Anxiolytic actions of chlordiazepoxide determine its effects on hyponeophagia in rats. Psychopharmacology (Berlin). 82:343–347; 1984. 38. Silver, W. L.; Mason, J. R.; Marshall, D. A.; Maruniak, J. A.: Rat trigeminal, olfactory and taste response after capsaicin desensitization. Brain Res. 333:45–54; 1985. 39. Smith, G. P.; Greenberg, D.; Corp, E.; Gibbs, J.: Afferent information in the control of eating. In: Obesity: Towards a molecular approach. New York: Alan R. Liss; 1990:63–79. 40. South, E.; Ritter, R. C.: Overconsumption of preferred food following capsaicin-pretreatment of the area postrema and adjacent nucleus of the solitary tract. Brain. Res. 288:243–251; 1983. 41. Swithers, S. E.; Hall, W. G.: Does oral experiences terminate ingestion? Appetite 23:113–138; 1984.
KELLY, CHAVEZ AND BERTHOUD 42. Tordoff, M. G.; Friedman, M. I.: Hepatic portal glucose infusions decrease food intake and increase food preference. Am. J. Physiol. 251: R192–R196; 1986. 43. VanVort, W.; Smith, G. P.: The relationship between the positive reinforcing and satiating effects of a meal in the rat. Physiol. Behav. 30:279–284; 1983. 44. Wiepkema, P. R.: Positive feedback at work during feeding. Behavior 39:266–272; 1971. 45. Young, A. M.; Woods, J. H.: Limitations on the antagonistic actions of opioid antagonists. Fed. Proc. 41:2333–2338; 1982. 46. Zittel, T. T.; Rothenhöfer, I.; Meyer, J. H.; Raybould, H. E.: Small-intestinal capsaicin-sensitive afferents mediate feedback inhibition of gastric emptying in rats. Am. J. Physiol. 267:G1142– G1145; 1994.
PII S0031-9384(98)00237-6
Transient Overconsumption of Novel Foods by Deafferentated Rats: Effects of Novel Diet Composition LISA A. KELLY, MARK CHAVEZ AND HANS-RUDOLF BERTHOUD1 Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Baton Rouge, LA, 70808 Received 11 November 1997; Accepted 6 August 1998 KELLY, L. A., M. CHAVEZ AND H.-R. BERTHOUD. Transient overconsumption of novel foods by deafferentated rats: Effects of novel diet consumption. PHYSIOL BEHAV 65(4/5) 793–800, 1999.—We recently demonstrated that capsaicin-treated rats consume more of an unfamiliar high-fat diet than vehicle-treated controls, but only on initial exposure (Chavez et al, 1997). We hypothesized that negative feedback signals carried by capsaicin-sensitive visceral afferents are critical for the regulation of intake of novel foods, but redundant pathways take over during subsequent exposures. To examine the role of nutrient content of the novel diet, rats were systemically treated with capsaicin (n 5 15) or vehicle (n 5 10), and exposed to 1) a fat/olestra diet that was isocaloric with chow; 2) a readily accepted fat-free cake; and 3) pure corn oil. Each 3-h feeding trial was preceded by 24-h food deprivation. Treated rats did not overconsume familiar chow, but did consume 50% more than controls of both the fat/olestra diet and the corn oil on first exposure; this suggests that capsaicin eliminated visceral afferents that normally carry satiety signals. However, the effect with the fat/olestra mixture was due primarily to depressed intake by controls, unlike the pure fat diets; this apparent neophobic response was blunted in treated rats. Because treated rats failed to overconsume the fat-free cakes, the neural system damaged by capsaicin appears to be linked to energy or fat sensory mechanisms, and possibly to hedonic responsiveness. © 1999 Elsevier Science Inc. Satiety Visceral afferents Food neophobia
High-fat diets
High carbohydrate diets
size and short-term food intake (4,25,30), it has not been possible to clearly demonstrate such a feeding effect with surgical or chemical ablation of vagal or visceral afferents (7,20,22). This failure is likely due to the extended recovery period necessary between ablation and testing, with the resulting development of compensatory satiety mechanisms; for this reason, a nontraumatic acute procedure would be ideal. We have further hypothesized that the salience of visceral afferent satiety signals may be minimal after a food has become familiar, because control of intake shifts to gustatory cues through learned associations. In support of this view, we have recently demonstrated that capsaicin-treated rats consume more of an unfamiliar high-fat diet than do vehicle-treated controls, and that this overconsumption occurs only on initial but not subsequent exposures (8). These findings are consistent with the idea that during ingestion of novel, but not familiar foods, negative feedback signals are carried by capsaicin-sensitive
THE consumption of food elicits a cascade of sensory signals as it is ingested, digested, absorbed, and metabolized. There is evidence for a number of vagal and spinal mechano- and chemosensors along the gastrointestinal tract and the portal hepatic axis; in addition, nutrients and their metabolites elicit the secretion of hormones and local messengers. It is thought that positive or reinforcing feedback signals are used by the brain to augment or maintain current ingestion (11,36,43), and negative feedback signals tend to suppress intake and accelerate the satiation process (14,16–18,33,39,41). In addition, there is increasing evidence that these various classes of sensory signals are used to form memorial representations and associations that guide future ingestion (3,10,12,15,23,27,42). Experimental support for these general concepts is still incomplete. For example, while it has been shown that blockade of vagal satiety pathways in the periphery by administration of selective CCK-A receptor antagonists can increase meal
1 To
Deprivation-induced feeding
whom requests for reprints should be addressed. E-mail: [email protected]
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visceral afferents critical for initial ingestion, but that redundant, capsaicin-resistant pathways take over this task during subsequent exposures. The novel test foods in our recent study were palatable, high-energy, high-fat foods, including a pure-fat diet and a complete liquid diet (Ensure), chosen specifically to make the new diet as different as possible from the maintenance diet. Thus, the aim of the present study was to determine which characteristics of the novel test food are necessary and sufficient to produce the overconsumption response in our experimental paradigm. For example, it may be essential that the test food have a higher energy density than the maintenance diet, or a different flavor, or mouth feel. South and Ritter (40) report that a highly palatable food (sucrose solution) must be used to observe intake differences between control, and either AP/NTS lesioned or fourth ventricular capsaicin-treated rats, while Curtis and Striker (9) demonstrated that rats treated with peripheral capsaicin also overconsumed a sucrose solution. Similarly, close examination of findings by Castonguay and Bellinger (7) reveal that capsaicin-treated rats initially overconsumed a novel sweetened milk diet, in comparison to control rats; in contrast, total intake and meal size of familiar chow was similar for the two groups. The role of macronutrient content in the induction of this behavioral effect is also unknown, as the fat content of both test diets in the initial study was much higher than that of the low fat chow maintenance diet. To address these issues of diet characteristics in the present study, vehicle- and capsaicin-treated rats maintained on chow were exposed to various novel foods after 24-h food deprivations. For Experiment 1, a pure fat/olestra mixture of the same caloric density as chow was used as the novel diet. In Experiment 2, the same rats were exposed to fat-free, highcarbohydrate cakes as the novel test diet to determine if the differential feeding response is retained with an unfamiliar food that is composed of carbohydrate rather than fat. MATERIALS AND METHODS
Animals Twenty-five male Sprague–Dawley rats (Harlan Industries, Indianapolis, IN) were used, weighing 180–250 g at the time of capsaicin- or vehicle-treatment. The animals were housed individually in hanging wire-mesh cages under standard laboratory conditions (12:12-h lighting schedule, lights on at 0700 h, 22638C), and 5001 Purina lab chow and tap water available ad lib except as noted prior to tests. All testing was conducted in the light phase between 1100 and 1400 h. Capsaicin Treatment Rats were treated consecutively with increasing doses of capsaicin. On each of 3 days, rats were injected under inhalation anesthesia (isoflurane) with either vehicle as a control, or a dose of capsaicin (12.5, 30, and 75 mg/kg, i.p., Sigma Chemicals, 98% grade). Capsaicin was dissolved freshly in a mixture of Tween 80 (10%), ethanol (10%), and sterile saline (80%) at the specific concentration. Following the first injection all rats exhibited respiratory arrest of between 1 and 5 min. In most cases, assistance by manually massaging the chest was sufficient to resume spontaneous breathing. In the remaining cases artificial respiration with oxygen was applied. One rat died during the first injection. During subsequent injections with the higher doses artificial respiration was rarely necessary.
Eye-Wipe Test Ten days following capsaicin treatment, one drop of 1% NH4OH was applied to the left eye with a Pasteur pipette and the number of eye wipes in 30 s and the latency to the first wipe were recorded. All capsaicin-treated rats fulfilled the criterion of less than three wipes and a latency of greater than 5 s to the first wipe. All vehicle control animals wiped vigorously, with a latency of less than 1 s and greater than 15 wipes/30 s. Short-Term Feeding Tests Begun 6 weeks after capsaicin treatment, Experiment 1 consisted of a series of five short-term feeding tests, performed at intervals of 6 days, each preceded by 24 h of food deprivation. The first test was conducted with the familiar pelleted chow diet, and the remaining four trials with a mixture of vegetable shortening (Crisco) and Olestra (a sucrose polyester, Proctor & Gamble) at a ratio of 1:1 by weight, resulting in a solid greasy diet mimicking the mouth feel of pure shortening, and isocaloric with the maintenance chow diet (3.4 kcal/g). On the day before the first test with the new diet, a small amount (,0.5 g) of the new diet was offered in an attempt to reduce any potential neophobic response to the food. The test food was given at 1100 h for a 3-h period, then chow was returned to the animals 5 h later at dark onset and overnight intake measured. During each trial, intake was measured at 30, 60, 120, and 180 min from the first bout of ingestion by weighing the food jar and spillage. Olestra has been known to cause soft stools, and because we noted that numerous rats of both groups showed soft feces after the first exposure to fat/olestra, the 24-h total wet weight of feces was measured for every trial afterward. Soft stool is a possible indicator of gastrointestinal comfort produced by Olestra. Individual cardboard spill pads were placed beneath each wire cage to collect the feces, and weighed immediately at the conclusion of the 24-h period. Soft feces were defined as any stool that did not appear as a firm pellet on visual inspection. Experiment 2 consisted of a second series of similar shortterm feeding trials using the same animals after a period of 4 weeks of ad lib access to chow. Following an initial test with chow, two tests with a commercially available fat-free dessert cake (Entenmann’s, 91.4% carbohydrate, 8.6% protein, 2.5 kcal/g) was used as the novel diet, because it has been reported that rats readily consume this food (35). Because no overconsumption response in capsaicin-treated rats was found with this novel food, a final test with pure corn oil (8.5 kcal/ mL) as the unfamiliar food was carried out. A comparison of energy density and macronutrient composition of the various diets is presented in Table 1. CCK Intake-Suppression Test After completion of all feeding tests, approximately 23 weeks following capsaicin treatment, animals were tested for the known food intake-suppression effects of exogenous CCK. In overnight food-deprived capsaicin- and vehicletreated rats, CCK (6 mg/kg, i.p.) or saline as a control, was administered 30 min before access to chow, and 1-h intake was measured. Half the animals of each group received CCK first, the other half received saline first. Data Analysis and Statistical Procedures The cumulative 3-h food intake results for each of the trials were analyzed independently, using a mixed-model ANOVA approach with treatment and time as factors. Comparisons of
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TABLE 1 ENERGY DENSITY AND MACRONUTRIENT COMPOSITION OF VARIOUS DIETS USED Novel Diets High-Fat
Energy density (kcal/g) % energy from fat % energy from COH % energy from protein
Fat-Free
Maintenance Chow Diet
Isocaloric to Chow (Olestra)
Corn Oil
Cakes
3.4 12 62 26
3.4 100 0 0
8.5 100 0 0
2.3 0 91 9
intake between chow trials and novel diet trials were also analyzed by a mixed-model ANOVA, with treatment as a fixed effect, time as a random effect, and trial as a repeated effect. When a significant effect of treatment or an interaction was found, Bonferroni t-tests were used in the post hoc pairwise analyses to isolate the effect. Results of the CCK test were analyzed using paired t-tests. RESULTS
Experiment 1 Capsaicin-treated rats weighed approximately 20 g less than the vehicle-controls 2 weeks following treatment. This initial small but significant weight difference was then maintained throughout the duration of the study, as both groups gained weight at a similar rate. Such a lasting difference in body weight was not observed in our earlier report and other experimental cohorts. During the first 3-h test with the familiar chow diet, capsaicin- and vehicle-treated rats consumed similar amounts (Fig. 1). In contrast, on the first trial with the new fat/olestra diet, capsaicin-treated rats consumed significantly more than controls, F(1, 19) 5 8.56, p , 0.01. Follow-up Bonferroni’s tests revealed that the treated rats consumed significantly more at both 60 and 120 min (pls , 0.05). If intake is compared to that of the preceding chow trial, it can be seen that the control rats showed depressed intake of the new diet, F(1, 246) 5 45.83,
p , 0.001, while the capsaicin-treated rats consumed similar amounts of chow and fat/olestra. Because it could be argued that the lower body weight of the capsaicin-treated rats may be responsible for differences in the short-term food intake trials, we analyzed 1-h food intake of the two groups with an adjustment for body weight. Using a model that allows different variances for the two treatment groups and Satterthwaite’s approximation of degrees of freedom, the analysis yielded a significant difference in 1-h food intake due to treatment, F(16, 8) 5 9.88, p , 0.01. Therefore, the overconsumption of olestra/fat diet by capsaicin-treated rats in the first trial was not due to differences in body weight. During the second exposure to the new diet, 6 days later, the consumption levels were reversed, such that the capsaicin treated rats now consumed less than the controls, F(1, 19) 5 10.21, p , 0.01. Post hoc comparisons revealed that intakes were significantly different at 1, 2, and 3 h. When compared with the consumption during the first exposure to the new diet, it can be seen that the control rats increased their intake, while that of the treatment group decreased. Despite this lower olestra intake, the capsaicin-treated rats showed a significantly higher wet weight of fecal output (22 vs. 16 g; p , 0.05; Fig. 2), and a higher incidence of soft feces (84% in treatment group vs. 40% in control group). In contrast, measurement of 24-h feces output during the second chow trial showed no difference between the groups in either feces weight or appearance. Olestra is well known to induce soft
FIG. 1. Mean (6SEM) 24-h deprivation-induced cumulative intake of chow (left panel) and solid fat/olestra (first and third exposures) in a series of short-term feeding trials. Vehicle rats (n 5 10) are represented by the solid line, and capsaicin-treated (n 5 13) by the broken line. Maintenance chow was returned 5 h after the conclusion of the test, at dark onset; trials were separated by 6 days. *p , 0.05 (Bonferroni’s correction).
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KELLY, CHAVEZ AND BERTHOUD stools (2,26), but our results further indicate that the bowel response to the fat substitute was aggravated in capsaicintreated rats. Capsaicin-treated rats continued to significantly underconsume the olestra diet during a third, F(1, 19) 5 10.21, p , 0.01, and fourth, F(1, 19) 5 16.89, p , 0.01, trial. In these two additional trials, the pattern of intake for both groups was similar to the second trial shown in Fig.1. Experiment 2
FIG. 2. Each bar represents mean (6SEM) 24-h fecal output after a short-term feeding trial: second exposure to fat/olestra, paired bars on the left, and chow, pair on the right. Vehicle rats (n 5 10) are represented by the left-hand bar and capsaicin-treated (n 5 13) by the right-hand bar. The lower, striped portion of each bar indicates the fecal output during the 3-h test period, while the upper solid portion shows the output during the remaining 21 h. Feces were collected on individual spill pads placed beneath the wire cage housing each rat, and weighed immediately at the conclusion of each period. *p , 0.05 (Bonferroni’s correction).
As in Experiment 1, the capsaicin-treated and control rats consumed almost identical amounts of chow, with similar time courses (Fig. 3). However, in contrast to Experiment 1 with the high-fat diet, capsaicin-treated rats did not overconsume the fat-free high-carbohydrate cakes on first exposure; rather, there was a small but significant treatment effect on food intake, F(1, 16) 5 6.17, p , 0.05, indicating higher consumption by controls, but with no individual time point showing a significant difference. Compared with the chow trial, both groups of rats consumed significantly more calories of the fatfree cakes, F(1, 140) 5 14.31, p , 0.001; F(1, 140) 5 61.61, p , 0.001. The second trial with this new food showed essentially the same results, with no significant difference in intake either between the groups or between the trials. During a final trial, which exposed the rats to pure corn oil 6 days after the last trial with fat-free cakes, the capsaicintreated rats overconsumed relative to both the controls, F(1, 18) 5 15.58, p , 0.001, and their own previous chow consumption, F(1, 140) 5 155, p , 0.001. Unlike the fat/olestra
FIG. 3. Mean (6SEM) 24-h deprivation-induced cumulative intake of chow (upper left panel), fat-free cake crumbs (Entenmann’s brand), and pure corn oil in consecutive short-term feeding trials. Vehicle rats (n 5 10) are represented by the solid line, and capsaicin-treated (n 5 11) by the broken line. Maintenance chow was returned 5 h after the conclusion of the test, at dark onset; trials were separated by 6 days. *p , 0.05 (Bonferroni’s correction).
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trial, the energy intake of control rats was not depressed relative to chow levels; on the contrary, it was slightly but significantly higher, F(1, 146) 5 6.97, p , 0.01. The patterns of intake were different between the two groups, as the treated rats exhibited essentially all consumption within the first 30 min, whereas the control group continued to consume corn oil throughout the 3-h trial.
Because the effect reappeared on exposure to the corn oil 6 days later, the loss cannot be attributed to experimental history. However, it remains to be clearly demonstrated that it is the macronutrient content in the cake that caused the loss of the overconsumption effect, rather than either energy density or palatability. For example, we have shown that the largest, absolute overconsumption (with energy intake larger than that of chow) occurs with high energy diets [solid fat (8) and corn oil (present results), of about 8 kcal/g], while the medium energy diets—Ensure (8) and fat/olestra (present results)— induce only a relative overconsumption that results from depressed intake by the control rats. Because the fat-free cakes with the lowest energy density of 2.3 kcal/g failed to induce any overconsumption, one might argue that high-energy density rather than fat is needed for the novel diet to cause overconsumption in capsaicin-treated rats. However, we have recently found that capsaicin-treated rats overconsume corn oil emulsion with a low-energy density of 0.8 kcal/mL by 70% (personal observations). Therefore, the lack of overconsumption of the fat free cakes is not likely due to the low energy content of this novel diet. Furthermore, the idea that high energy content of the novel diet is an important prerequisite for absolute overconsumption to occur does not appear to be supported by the results of another recent study, where 10% sucrose (0.45 kcal/mL) was consistently overconsumed by capsaicin-treated rats (8). South and Ritter (40) also reported that a sucrose solution induced overconsumption in a model with fourth ventricular capsaicin administration. Although we have not yet tested sucrose solutions for overconsumption in our model, we have no reason to assume that it would not be effective, given that Curtis and Stricker (9) used the same capsaicin treatment and the same strain of rats. Thus, the question becomes: why did solid carbohydrate not induce overconsumption, while the sucrose solution did? As it is well recognized that hydration of carbohydrate renders it extremely palatable to rats (29), it is possible that a solid form of carbohydrate cannot induce the overconsumption response due to lesser palatability than either sucrose solutions or fat-based diets. One useful approach in dissection of the role of macronutrients in the overconsumption response would be to isolate the effects of taste or palatability from those of the postingestive consequences. For example, duodenal preloads of carbohydrate or fat could be given prior to measuring food deprivation-induced intake of a novel food to directly determine whether capsaicin-treatment differentially interferes with the generation of negative feedback from each macronutrient.
CCK-8 Feeding Suppression Test CCK-8 suppressed intake of chow in the overnight fooddeprived rats by about 50% (t 5 8.9, p , 0.05; Fig. 4) in vehicle-treated control rats, but failed to induce significant suppression in capsaicin-treated rats (t 5 1.78, p 5 0.11). DISCUSSION
Similar to the results of our previous report with a novel high-energy diet of pure vegetable shortening (8), capsaicintreated rats overconsumed, relative to vehicle-treated controls, two other novel high-fat diets on first exposure: a fat/ olestra mixture and pure corn oil. In contrast, an unfamiliar, fat-free high carbohydrate food was not overconsumed. These results could be interpreted as evidence that fat is a necessary component for a novel food to induce an overingestion response in capsaicin-treated rats. Because systemic capsaicin treatment is known to destroy a population of fine, unmyelinated visceral afferents (1,19), the phenomenon of overingestion might then best be explained by the loss of negative feedback signals from ingested fat that normally suppress further intake in the intact animal. In addition, the fact that the overconsumption is transient, as it is only observed during the first 3-h exposure to the novel diet, suggests that the animal does not exclusively rely on this capsaicin-sensitive mechanism, but uses redundant pathways to recalibrate future intake of the new diet. However, before we can adopt such a simple interpretation of the results, several issues must be addressed. Role of Diet Composition In contrast to the results with the novel fat food in Experiment 1, and our previous findings with vegetable shortening and Ensure, the capsaicin-treated rats did not overconsume the novel fat-free, high-carbohydrate cakes on first exposure.
Does Capsaicin Treatment Interfere With the Expression of Normal Food Neophobia?
FIG. 4. Effect of exogenous CCK (6 mg/kg, i.p, striped bars) or saline (solid bars) on 30-min chow intake of vehicle (n 5 10) and capsaicin-treated (n 5 13) rats. Overnight food-deprived rats were injected 30 min prior to access to food. Note that food intake suppression by CCK is completely abolished in capsaicin-treated rats. Tests were conducted 23 weeks following capsaicin treatment. *p , 0.01 (t-test).
Because the phenomenon of relative overconsumption, as reported here, is dependent on decreased intake by the control rats, our hypothesis of blunted negative feedback is insufficient to account for this. Therefore, we propose two additional mechanisms that may also contribute to the differential intake levels observed between treated and control rat. First, it appears that capsaicin treatment interferes with the normal expression of food neophobia. It is well established that most novel foods are cautiously approached by rats (6). Before any oral sampling, the smell of food can already be recognized as novel, so that the animal may not touch the food for a considerable time. Eventually, small amounts are ingested for the purpose of detecting any adverse postingestive effects. Although there is an interaction of this caution
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with the level of hunger (37), 24-h food deprivation is not sufficient to overcome it. If capsaicin treatment decreased sensitivity to smell and taste, this could explain the apparent interference with neophobic hypophagia. It has been reported that capsaicin treatment does not cause a functional loss in tasting ability (38), but the authors concurrently observed that the compound action potential recorded from the olfactory nerve in response to various odor stimuli was decreased. Given that postingestive consequences must be experienced to overcome neophobia (5,28), it is unlikely that the capsaicin-induced damage to postingestive sensory mechanisms resulted in a quicker attenuation of gustatory neophobia; rather, it is more plausible that capsaicin treatment interferes with either smell or taste during the active phase of neophobia, or with brain stem mechanisms that translate neophobia into the inhibition of food intake. In light of this, it is possible that the fat-free cake failed to induce differential consumption by the treated rats because it did not provoke a neophobic response in the control rats.
15 min, these authors concluded that it is the attenuation of early signals of gastric distension that induces overingestion by treated rats. These opposing interpretations may be reconciled with the proposal that low and high threshold subpopulations of gastric distension afferents are differentially sensitive to capsaicin, and that a population of low-threshold fibers may be most susceptible to the neurotoxin. Because the above-cited studies demonstrating capsaicin-resistant vagal fibers used only relatively large distension volumes, the results, therefore, cannot rule out the existence of capsaicin-sensitive low-threshold fibers. That relatively small volumes of gastric distension can suppress intake in intact rats has also recently been demonstrated by Davis and co-workers (13). Furthermore, in addition to the loss of a directly inhibitory effect on intake via low-threshold gastric distension afferents, more distal vagal receptors could also indirectly affect intake via slowing of gastric emptying. There is evidence that small intestinal capsaicin-sensitive afferents mediate feedback inhibition of gastric emptying (46).
Does Capsaicin Treatment Enhance the Positive or Reinforcing Aspects of Foods?
Implications for Learning and Conditioned Satiety
It has been suggested that meal size, and thus, short-term control of food intake is determined by balance between positive and negative feedback signals (39). Therefore, overingestion by capsaicin-treated rats could be the result of either decreased negative feedback, as earlier hypothesized, or else increased positive feedback. This, in turn, implies that the treatment may result in the amplification of positive or reinforcing properties of food (12,24,44). A more precise examination of the microstructure of the pattern of overconsumption, either with a variety of test diets, or with a highly palatable maintenance food, is one important step in discriminating between these mechanisms underlying the overconsumption. In addition, opioid receptor dependency of the overconsumption effect should be tested in future experiments, as it is known that opioid receptors are involved in the mediation of reinforcing or rewarding properties of foods (21,45). What Is the Neurological Substrate Damaged by Capsaicin Treatment and What Portion of This Substrate Is Responsible for Overconsumption? From a more functional point of view, the above discussion implies that capsaicin damage may have impinged on a number of neural pathways, not necessarily limited to primary afferent fibers. Anatomical studies show that systemic capsaicin treatment in adult rats leads to degeneration of a distinct population of visceral afferents, including but not limited to those of dorsal root and nodose-vagal origin (1,19,34). In contrast, another population of fine unmyelinated fibers of at least vagal origin and innervating primarily the upper gastrointestinal tract seems to remain both physically and functionally intact (1,32). Specifically, it has been shown by various methods, including anatomical tracing, c-fos expression, and electrical unit recording (1,32), that many vagal afferents sensitive to gastric distension are capsaicin resistant, so that there may be only partial loss of negative feedback signals from gastric distension receptors in capsaicin-treated rats. This also suggests that the loss of signals from duodenal nutrient sensors may be the more likely explanation for their overingestion of novel foods. This conclusion, however, is in direct contrast to the one reached by Curtis and Stricker (9); based on their findings that capsaicin-treated rats overconsumed a variety of solutions, including plain tap water, and did so within the first 5 to
One striking finding from both our current and previous studies is the consistent loss of the overconsumption effect by the second and third trials with the novel food. In Experiment 1 of the present study, this could have been due to malabsorption of the fat/olestra diet, as indicated by the soft feces, which appeared to be aggravated by capsaicin treatment. The resulting gastrointestinal malaise could have produced a mild taste aversion. Similarly, in our previous study, the capsaicintreated rats ingested so much of the vegetable shortening that they may have experienced indigestion, thus producing a mild aversion during the next trials that could have interfered with possible overconsumption. However, such arguments are harder to make for one of the experiments reported earlier (8), using the nutritionally complete liquid diet Ensure as the novel food, for which there was also no sign of overingestion during the second and third trials. Furthermore, in support of our finding, closer inspection of data shown by Castonguay and Bellinger (7) indicates that while capsaicin-treated rats indeed consumed more of a novel milk diet than controls on first exposure, intakes were almost identical during the third daily 30-min exposure. We suggest that the initial overconsumption seen with capsaicin-treated rats disappears on repeated exposure due to postingestive consequences. We further believe that these capsaicin-resistant signals are also the reason that both capsaicin-treated rats do not overconsume the familiar chow in our studies, and that Castonguay and Bellinger (7) did not detect long-term changes in meal size with the chow diet. In contrast to this transient character of the overingestive response in our experimental setting, as well as that of Castonguay and Bellinger (7), Curtis and Stricker (9) observed that their capsaicin-treated rats still significantly overconsumed sucrose during the third daily 30-min test. However, procedural differences between our study and theirs may explain the apparent discrepancy. Although our paradigm uses 24-h food deprivation from a maintenance chow diet, Curtis and Striker use a restricted feeding schedule with 10 g of chow per day, and daily access of only 30 min to the sucrose solution, after 2 days continuous access. These procedures may have created considerable differences in the levels of hunger and nutritional expectation before presentation of the test food, which interfered with the recalibration process. Thus, it is possible that it may simply take more than three 30-min ex-
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posures within this paradigm for recalibration to occur. Alternatively, it is also possible that the low-energy density of the 10% sucrose induced only weak postingestive consequences, which were unable to modify subsequent intake in capsaicintreated animals. It seems clear that capsaicin treatment permanently destroys a substantial number of several types of visceral afferents, as the anatomical evidence is supported by the finding that capsaicin treatment induces a permanent loss of the effectiveness of CCK to suppress intake (31). Thus, for treated rats to exhibit such eventual control of short-term intake as we and Castonguay and Bellinger (7) report, there must exist a redundant satiety system. Furthermore, given this reasoning, the redundant pathways must not be capsaicin sensitive. In
fact, the existence of capsaicin-resistant, probably nonvagal, sensory pathways from the alimentary canal to the brain has been demonstrated by Lucas and Sclafani (23), who showed that capsaicin treatment did not interfere with the development of flavor preferences due to duodenal infusions. Thus, these pathways represent the prime candidates for functionally replacing other visceral efferents damaged by capsaicin. ACKNOWLEDGEMENTS
We would like to thank Dr. Julia Volaufova for advice and help on statistical analysis. This work was partially supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-47348 to H.-R.B., and DK-32089 to G.A.Bray).
REFERENCES 1. Berthoud, H.-R.; Patterson, L. M.; Willing, A. E.; Mueller, K.; Neuhuber, W. L.: Capsaicin-resistant vagal afferent fibers in the rat gastrointestinal tract: Anatomical identification and functional integrity. Brain Res. 746:195–206; 1997. 2. Blackburn, H.: Olestra and the FDA. N. Engl. J. Med. 334:984– 986; 1996. 3. Booth, D. A.: Conditioned satiety in the rat. J. Comp. Neurol. 81:457–471; 1972. 4. Brenner, L.; Ritter, R.C.: Peptide cholesystokinin receptor antagonist increases food intake in rats. Appetite 24:1–9; 1995. 5. Buro sˇ ová, O; Bure sˇ , J.: Post-ingestive interference with brain function prevents attenuation of neophobia in rats. Behav. Brain Res. 1:299–312; 1980. 6. Carroll, M. E.; Dinc, H. I.; Levy, C. J.; Smith, J. C.: Demonstrations of neophobia and enhanced neophobia in the albino rat. J. Comp. Physiol. Psychol. 89:457–467; 1975. 7. Castonguay, T. W.; Bellinger, L. L.: Capsaicin and its effects upon meal patterns, and glucagon and epinephrine suppression of food intake. Physiol. Behav. 40:337–342; 1987. 8. Chavez, M.; Kelly, L.; York, D. A.; Berthoud, H.-R.: Chemical lesion of visceral afferents causes transient overconsumption of unfamiliar high-fat diets in rats. Am. J. Physiol. 272:R1657– R1663; 1997. 9. Curtis, K. S.; Stricker, E. M.: Enhanced fluid intake by rats after capsaicin treatment. Am. J. Physiol. 272:R704–R709; 1997. 10. Davidson, T. L.; Altizer, A. M.; Benoit, S. C.; Walls, E. K.; Powley, T. L.: Encoding and selective activation of “metabolic memories” in the rat. Behav. Neurosci. 111:1–17; 1996. 11. Davis, J. D.; Levine, M. W.: A model for the control of ingestion. Psychol. Rev. 84:379–412; 1977. 12. Davis, J. D.; Perez, M. C.: The acquired control of ingestive behavior in the rat by flavour-associated post-ingestional stimulation. Physiol. Behav. 54:1221–1226; 1993. 13. Davis, J. D.; Smith, G. P.; Sayer, J. L.: Reduction of intake in the rat due to gastric filling. Am. J. Physiol. 272:R1599–R1605; 1997. 14. Deutsch, J. A.: The role of the stomach in eating. Am. J. Clin. Nutr. 42:1040–1043; 1985. 15. Deutsch, J. A.; Tabuena, J. A.: Learning of gastrointestinal satiety signals. Behav. Neural Biol. 45:292–299; 1986. 16. Friedman, M. I.: Control of energy intake by energy metabolism. Am. J. Clin. Nutr. (Suppl.) 62:1096S–1100S; 1995. 17. Geary, N.; Le Sauter, J.; Unkyung, N.: Glucagon acts in the liver to control spontaneous meal size in rats. Am. J. Physiol. 33:R116– R122; 1993. 18. Greenberg, D.: Intestinal satiety. In: Smith, G. P.; Gibbs, J., eds. Structure and mechanisms of satiety. London: Oxford University Press; 1996. 19. Holzer, P.: Capsaicin: Cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol. Rev. 43:143–172; 1991. 20. Kraly, F. S.; Jerome, C.; Smith, G. P.: Specific postoperative syn-
21. 22. 23. 24. 25. 26.
27. 28. 29. 30. 31. 32. 33.
34.
35. 36.
dromes after total and selective vagotomies in the rat. Appetite 7:1–17; 1986. Levine, A. S.; Weldon, D. T.; Grace, M.; Cleary, J. P.; Billington, C. J.: Naloxone blocks that portion of feeding driven by sweet taste in food-restricted animals. Am. J. Physiol. 37:R248–R252; 1995. Lorenz, D. N.: Effects of gastric filling and vagotomy on ingestion, nipple attachment and weight gain by suckling rats. Dev. Psychobiol. 16:469–483; 1983. Lucas, F.; Sclafani, A.: Capsaicin attenuates feeding suppression but not reinforcement by intestinal nutrients. Am. J. Physiol. 270:R1059–R1064; 1996. Lucas, G. A.; Timberlake, W.: Interpellet delay and meal pattern in the rat. Physiol. Behav 43:259–264; 1988. Meisner, J. A.; Smith, G. P.; Gibbs, J.; Tyrka, A.: Intravenous infusion of CCK-A receptor antagonist increases food intake in rats. Am. J. Physiol. 262:R216–R219; 1992. Mellies, M. J.; Jandacek, R. J.; Taulbee, J. D.; Tewksbury, M. B.; Lamkin, G.; Baehler, L.; King, P.; Boggs, D.; Goldman, S.; Gouge, A.; Tsang, R.; Glueck, C. J.: A double-blind, placebocontrolled study of sucrose polyester in hypercholesterolemic outpatients. Am. J. Clin. Nutr. 37:339–346; 1983. Mook, D. G.: Oral factors in appetite and satiety. Ann. NY Acad. Sci. 575:265–278; 1989. Pelchet, M. L.: What is “mere exposure”? Proceedings of the Annual Meeting of the Society for the Study of Ingestive Behavior, Baltimore, MD; 1997. Ramirez, I.: Feeding a liquid diet increases energy intake, weight gain and body fat in rats. J. Nutr. 117:2127–2134; 1987. Reidelberger, R. D.; O’Rourke, M. F.: Potent cholecystokinin antagonist L 364718 stimulates food intake in rats. Am. J. Physiol. 257:R1512–R1518; 1989. Ritter, R. C.; Ladenheim, E. E.: Capsaicin pretreatment attenuates suppression of food intake by cholecystokinin. Am. J. Physiol. 248:R501–R504; 1985. Ritter, R. C.; Ritter, S.; Ewart, W. E.; Wingate, D. L.: Capsaicin attenuates hindbrain neuron responses to circulating cholecystokinin. Am. J. Physiol. 257:R1162–R1168; 1989. Ritter, R. C.; Brenner, L.; Yox, D. P.: Participation of vagal sensory neurons in putative satiety signals from the upper gastrointestinal tract. In: Ritter, S.; Ritter, R. C.; Barnes, C. D., eds. Neuroanatomy and physiology of abdominal vagal afferents. Boca Raton, FL: CRC Press; 1992:221–248. Ritter, S.; Dinh, T. T.: Capsaicin-induced neuronal degeneration; Silver impregnation of cell bodies, axons, and terminals in the central nervous system of the adult rat. J. Comp. Neurol. 271:79– 90; 1988. Sclafani, A.; Weiss, K.; Cardieri, C.; Ackroff, K.: Feeding response of rats to no-fat and high-fat cakes. Obesity Res. 1:173– 178; 1995. Sclafani, A.: How food preferences are learned: Laboratory animal models. Proc. Nutr. Soc. 54:419–427; 1995.
800 37. Shephard, R. A.; Estall, L. B.: Anxiolytic actions of chlordiazepoxide determine its effects on hyponeophagia in rats. Psychopharmacology (Berlin). 82:343–347; 1984. 38. Silver, W. L.; Mason, J. R.; Marshall, D. A.; Maruniak, J. A.: Rat trigeminal, olfactory and taste response after capsaicin desensitization. Brain Res. 333:45–54; 1985. 39. Smith, G. P.; Greenberg, D.; Corp, E.; Gibbs, J.: Afferent information in the control of eating. In: Obesity: Towards a molecular approach. New York: Alan R. Liss; 1990:63–79. 40. South, E.; Ritter, R. C.: Overconsumption of preferred food following capsaicin-pretreatment of the area postrema and adjacent nucleus of the solitary tract. Brain. Res. 288:243–251; 1983. 41. Swithers, S. E.; Hall, W. G.: Does oral experiences terminate ingestion? Appetite 23:113–138; 1984.
KELLY, CHAVEZ AND BERTHOUD 42. Tordoff, M. G.; Friedman, M. I.: Hepatic portal glucose infusions decrease food intake and increase food preference. Am. J. Physiol. 251: R192–R196; 1986. 43. VanVort, W.; Smith, G. P.: The relationship between the positive reinforcing and satiating effects of a meal in the rat. Physiol. Behav. 30:279–284; 1983. 44. Wiepkema, P. R.: Positive feedback at work during feeding. Behavior 39:266–272; 1971. 45. Young, A. M.; Woods, J. H.: Limitations on the antagonistic actions of opioid antagonists. Fed. Proc. 41:2333–2338; 1982. 46. Zittel, T. T.; Rothenhöfer, I.; Meyer, J. H.; Raybould, H. E.: Small-intestinal capsaicin-sensitive afferents mediate feedback inhibition of gastric emptying in rats. Am. J. Physiol. 267:G1142– G1145; 1994.