- Email: [email protected]
Two effects of high-fat diets on the satiating potency of cholecystokinin-8
Physiology & Behavior 78 (2003) 19 – 25
Two effects of high-fat diets on the satiating potency of cholecystokinin-8 Ann-Marie Torregrossa, Gerard P. Smith* E.W. Bourne Laboratory, Department of Psychiatry, Weill Medical College of Cornell University, New York, NY, USA E.W. Bourne Laboratory, New York-Presbyterian Hospital, Westchester Division, 21 Bloomingdale Road, White Plains, NY 10605, USA Received 6 February 2002; received in revised form 1 July 2002; accepted 6 August 2002
Abstract Chronic ingestion of diets containing 34% or 54% fat have been reported [Peptides 19 (1998) 1407] to decrease the inhibitory effect on food intake of doses of cholecystokinin-8 (CCK-8) less than 1 mg/kg ip. We attempted to replicate this phenomenon in three experiments by comparing the effect of high-fat and low-fat diets on the threshold dose of CCK-8 for inhibition and on the dose – response function for doses of CCK-8 that ranged from 0.125 to 2.0 mg/kg. The first experiment tested rats five times per week. Rats on a 34% fat diet had a higher threshold (1.0 mg/kg) than rats on a 5% fat diet (0.25 mg/kg). The dose – response functions, however, were not significantly different. This result replicated the earlier report [Peptides 19 (1998) 1407]. The second experiment tested rats maintained on the same diets every other day as in the original report. It failed, however, to replicate the results of the first experiment or the earlier report because the threshold doses and the dose – response functions of CCK-8 were not significantly different between rats on 34% and 5% fat diets. The third experiment compared the potency of CCK-8 in rats on a 60% fat diet with the potency in rats on a 5% fat diet. CCK-8 was significantly more potent in the rats on the 60% fat diet because the threshold dose of these rats was lower (0.125 mg/kg) than in rats on the 5% fat diet (0.25 mg/kg), and the dose – response function in rats on the 60% fat diet was significantly more potent than in rats on the 5% fat diet. Thus, we observed two effects of the chronic ingestion of high-fat diets on the inhibitory potency of CCK-8: (1) A 34% fat diet increased the threshold dose of CCK-8 without changing the dose – response function in one of two experiments. (2) A 60% fat diet increased the potency of CCK-8 because it decreased the threshold dose and increased the dose – response function significantly. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Control of food intake; Gut peptides; Cholecystokinin-33; Diet-induced obesity; Fat pads; Satiation
1. Introduction In 1998, Covasa and Ritter [1] reported that low doses of cholecystokinin-8 (CCK-8) administered intraperitoneally decreased 30-min intake significantly less in rats maintained on a 34% fat diet than in rats maintained on a 5% fat diet. The 34% fat diet decreased the potency of doses of CCK-8 smaller than 1 mg/kg, but it had no effect on the inhibitory potency of 1 mg/kg CCK-8. Thus, the chronic ingestion of the 34% fat diet decreased the sensitivity of rats to the satiating potency of CCK-8, but it apparently did not shift the upper part of the dose – response function to the right. These experiments also demonstrated that the effect of the 34% fat diet depended on rats eating the diet for at least 2 weeks prior to the intake tests. The authors concluded that the effect of the 34% fat diet was not due to the fat content of * Corresponding author. E.W. Bourne Laboratory, New York-Presbyterian Hospital, Westchester Division, 21 Bloomingdale Road, White Plains, NY 10605, USA. Tel.: +1-914-997-5935; fax: +1-914-682-3793. E-mail address: [email protected] (G.P. Smith).
the diet that rats ate during the intake test, to the caloric density of the diet, or to a difference in the amount of body weight gained on the 5% and 34% diets. This interesting effect of a high-fat diet on the potency of low doses of exogenous CCK-8 was followed up in two experiments. First, CCK-8 (0.25 –2.0 mg/kg ip) decreased gastric emptying less in rats on a 54% fat diet than in rats on a 5% fat diet [2]. Second, CCK-8 (0.25 mg/kg ip) produced a significantly smaller number of cells with Fos-like immunoreactivity (Fos LI) in the dorsal vagal complex in rats on a 34% fat diet than in rats on a 5% fat diet [3]. These results with exogenous CCK-8 were apparently extended to endogenous CCK by using intestinal infusions of oleate that had been shown to decrease food intake by releasing endogenous CCK [4]. In comparison to low-fat diets, high-fat diets (34% or 54%) decreased the inhibitory effect of duodenal infusions of oleate on intake [5], gastric emptying [2], and the number of Fos LI cells in the dorsal vagal complex [6]. The parallel results of high-fat diets on the inhibitory potencies of exogenous CCK-8 and intraintestinal oleate
0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 8 8 8 - 0
20
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
were consistent with the possibility that the decreased response to oleate was due to the decreased response to endogenous CCK released by oleate [4]. No direct evidence, however, was presented for this causal relationship. In a recent experiment, Covasa et al. [7] demonstrated that when circulating CCK was increased chronically by continuous intraperitoneal infusion for 28 days, the potency of an intraperitoneal injection of CCK-8 was reduced. This result suggested that chronic elevation of CCK produced by chronic ingestion of a high-fat diet could be an important cause for the reduced potency of CCK-8 observed with that diet. The mechanism of the reduced response to CCK-8 or intestinal oleate is unknown. The mechanism could be peripheral, involving a reduction in receptor binding due to down-regulation of the number of receptors or a reduced transduction by vagal afferent neurons that mediate the satiating effect of CCK-8 and intraintestinal oleate. On the other hand, the mechanism could be central—the change in metabolic state produced by the chronic consumption of a high-fat diet could alter the central processing of the peripheral vagal afferent information. Given our long-standing interest in the satiating effect of exogenous and endogenous CCK [8], we decided to investigate the site and kind of mechanism underlying the effect of high-fat diets. We began by attempting to reproduce the original report of the effect of a 34% diet on the potency of CCK-8.
2. Experiment 1 2.1. Methods 2.1.1. General conditions Twelve male Sprague Dawley rats (Taconic Farms, Germantown, NY), weighing 337– 402 g at the beginning of the experiment, were housed individually in polycarbonate, solid-bottom cages (24 20 29 cm) with cob bedding. The lighting schedule was a 12-h, light/dark cycle; lights were on from 0700 to 1900 h. All rats had access to tap water ad libitum. Six rats (372.3 ± 9.5 g) were maintained on a 34% fat diet and 6 (373.5 ± 8.3 g) were maintained on a 5% fat diet. The preparation and constituents of the diets (Table 1) were identical to the diets used by Covasa and Ritter [1]. Note that the 5% fat diet is high in carbohydrate in the form of corn starch and that the 34% fat diets contain no carbohydrate. Diets were presented in spill-resistant metal bowls (Lab. Products, Seaford, DE) resting in larger ceramic bowls to collect any spillage. Rats were adapted to their assigned diet for at least 2 weeks prior to testing. 2.1.2. Test schedule Rats were tested daily (Monday – Friday). Diets were removed at 1630 h and weighed bowls of fresh diet were returned at 0930 h. Five minutes before the presentation of the diets, all rats received an intraperitoneal injection (1 ml/
Table 1 Diet constituents (wt.%)
5% fat
34% fat
60% fat
Casein Corn starch Fat Vitamin mix Mineral mix Alphacel DL-Methionine
20 65.4 5 2.2 4 3 0.4
20 0 34 2.2 4 39.4 0.4
20 0 60 2.2 4 13.4 0.4
The composition of the 5% and 34% fat diets are from Ref. [1]. The 60% fat diet was used in Experiment 3. The preparation of the diets was as described in Ref. [1] and in personal communication with Covasa and Ritter. Casein and methionine were purchased from Sigma (St. Louis, MO). Alphacel and corn starch were purchased from ICN Biomedicals (Aurora, OH). The fat mixture consisted of 1% Canola vegetable oil (Pure Wesson Canola Oil, Fullerton, CA) and 4% Lard (Armour Lard, Omaha, NE) and it was the one used by Covasa and Ritter (personal communication). The vitamin and mineral mixes were purchased from Bio-Serve (Frenchtown, NJ).
kg) of either CCK-8 (Peninsula Laboratories, Belmont, CA; 1.0, 0.5, 0.25, 0.125, and 2.0 mg/kg in that order) in 0.9% saline, equimolar doses of porcine CCK-33 (Peptides International, Louisville, KY; 3.3, 1.65, 0.83, 0.42, and 6.6 mg/kg in that order) in 0.9% of saline, or 0.9% saline (bacteriostatic 0.9% sodium chloride, Abbott Laboratories, North Chicago, IL). Each dose of both peptides was tested once. The tests with CCK-8 were completed before the tests with CCK-33 began. At least one test after an injection of 0.9% saline preceded a test in which CCK-8 or CCK-33 was injected. After 30-min access to the diet, the bowls of diet were weighed again and the difference in weight from the beginning of the test was the measure of intake because spillage did not occur during the tests. Body weights were recorded prior to each test. After the test on Friday, rats had access to their diets ad libitum until 1630 h on Sunday. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Weill Medical College of Cornell University. 2.1.3. Statistics Separate two-way analysis of variance with repeated measures on dose of peptide were used to analyze the effect of the two fat diets on the satiating potency of all of the doses of CCK-8 and CCK-33. We used planned comparisons by ttests to determine the smallest dose of CCK-8 or CCK-33 that inhibited intake compared to the mean intake after saline treatment in Experiments 1 and 2. Because the intakes after saline in Experiment 3 differed significantly over time ( P=.05), the intake after CCK-8 was compared to the intake after saline on the day that preceded that dose in that experiment. Given the extensive evidence that CCK-8 inhibits intake, but does not increase it, we considered that a difference between intakes after CCK-8 or CCK-33 and saline was significant when P=.05 or less, one-tailed. This estimate of the threshold dose for inhibition is a measure of the sensitivity that was reported to be changed by the high-fat diet [1].
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
21
The effects of fat diets on body weight gain were analyzed with t-tests, two-tailed. Differences were considered significant when P=.05 or less. All statistical analyses were performed using SYSTAT 7.0 statistical analysis software program and graphics were created with Sigma Plot for Windows 2.0 and 3.0. 2.2. Results The smallest dose of CCK-8 that inhibited intake significantly was 0.25 mg/kg in rats on the 5% fat diet ( P < .03) and 1 mg/kg in rats on the 34% fat diet ( P=.00). The smallest dose of CCK-33 that inhibited intake significantly was 0.83 mg/kg in rats on the 5% fat diet ( P=.02) and 1.65 mg/kg in rats on the 34% fat diet ( P < .01). Note that the equimolar threshold doses of CCK-33 and CCK-8 were the same on the 5% fat diet, but the threshold dose CCK-33 was smaller than that of CCK-8 in rats on the 34% fat diet. In contrast, the percentage of fat in the diet did not produce a significant difference in the satiating potency of CCK-8 [ F(1,10) = 2.28, P=.16; Fig. 1] or CCK-33 [ F(1,10) = 0.37, P=.56; Fig. 2]. There was, however, a significant effect of dose of CCK-8 [ F(5) = 17.76, P=.00] and dose of CCK-33 [ F(5)=37.75, P=.00] on the intakes of both diets, but no interaction between diet and dose of CCK-8 [ F(5,50) = 0.85, P=.52] or CCK-33 [ F(5,50) = 1.56, P=.19]. The diets did not produce a significant difference in weight gain from the beginning to the end of the experiment (5%, 105.7 ± 11.27 g; 34%, 93.7 ± 3.69 g; P=.35).
Fig. 2. Data are mean ± S.E. grams of diet eaten in 30 min. Empty columns are intakes of 5% fat diet (n = 6); black columns are intakes of 34% fat diet (n = 6). Doses of CCK-33 were equimolar to the doses of CCK-8 in Fig. 1. Intakes after the zero dose are the pooled intakes from the five tests after saline administration on the days that preceded CCK-33 tests. * Smallest dose of CCK-33 that produced a significantly smaller intake than the intake after saline, P < .05.
2.3. Discussion The threshold dose in rats on the 34% fat diet was four times as large for CCK-8 and twice as large for CCK-33 as the threshold dose in rats on the 5% fat diet. Thus, rats on the 34% fat diet were less sensitive to the satiating effect of CCK-8 and CCK-33 than rats on the 5% fat diet. The satiating potencies of all of the doses of CCK-8 and CCK-33 in rats ingesting the 34% fat diet, however, were not significantly different from their satiating potencies in rats ingesting the 5% fat diet. This combination of decreased sensitivity for low doses of CCK-8 and no change in the dose – response function is consistent with the results of Covasa and Ritter [1]. The same combination of results with CCK-33 extends the effect of 34% fat diets to a larger form of CCK. There was a difference in the test schedule between our protocol and that of Covasa and Ritter [1]. We tested our animals daily, Monday through Friday, while Covasa and Ritter [1] tested theirs every other day. Because the difference in test frequency might influence the results, we repeated the experiment using their protocol.
Fig. 1. Data are mean ± S.E. grams of diet eaten in 30 min. Empty columns are intakes of 5% fat diet (n = 6); black columns are intakes of 34% fat diet (n = 6). Intakes after the zero dose are the pooled intakes from the five tests after saline administration on the days that preceded CCK-8 tests. * Smallest dose of CCK-8 that produced a significantly smaller intake than the intake after saline, P < .05.
3. Experiment 2 There were three differences between this experiment and Experiment 1. One was the schedule of testing. In Experiment 1, rats were tested daily; in this experiment, they were
22
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
measure of intake. After the test on Friday, rats had access to the diets ad libitum until 1630 h on Sunday. Body weights of all rats were recorded daily. This occurred prior to the test on test days or at the same time on Tuesdays and Thursdays. Rats were sacrificed at the conclusion of the experiment and epididymal and inguinal fat pads were dissected out of the animals, blotted with filter paper, and weighed. 3.1.1. Statistics The effect of the fat diets on threshold dose for inhibition of intake, on the potency of all of the doses of CCK-8, and on body weight gain were analyzed exactly as in Experiment 1. The effect of fat diets on the weights of inguinal and epididymal fat pads were analyzed with t-tests, two-tailed. Differences were considered significant when P=.05 or less. 3.2. Results Fig. 3. Data are mean ± S.E. grams of diet eaten in 30 min in Experiment 2. Empty columns are intakes of 5% fat diet (n = 5); black columns are intakes of 34% fat diet (n = 9). * Smallest dose of CCK-8 that produced a significantly smaller intake than the intake after saline, P < .05.
tested every other day. The second difference is that we tested only CCK-8 because the results with CCK-8 and CCK-33 were similar in Experiment 1. The third difference is that we measured the weight of inguinal and epididymal fat pads at the end of the experiment to determine if the 34% fat diet increased the weight of these fat pads more than 5% fat diet even though the 34% fat diet had not increased body weight in Experiment 1 more than the 5% fat diet did. 3.1. Methods Fourteen male Sprague Dawley Rats (Taconic Farms), weighing 172– 222 g at the beginning of the experiment, were used. These rats had no prior experience with the test diets or with the test protocol. All housing, lighting conditions, and diets were as described in Experiment 1. Nine rats (197.0 ± 5.1 g) were maintained on the 34% fat diet and five rats (203.4 ± 8.5 g) were maintained on the 5% fat diet. All rats had tap water ad libitum. Rats were adapted to their assigned diet for at least 2 weeks prior to testing. Diet was presented in spill-resistant metal bowls resting in larger ceramic bowls on top of plastic trays (M. Covasa, personal communication). Animals were tested on Mondays, Wednesdays, and Fridays after being deprived of their diet at 1630 h on the prior day. At 0930 h, the food bowls were filled with fresh diet, weighed, and presented. Five minutes before presentation of the diets, rats received an intraperitoneal injection (1 ml/kg) of either CCK-8 (1.0, 0.5, 0.25, 0.125, and 2.0 mg/kg in that order) in saline or saline alone. After 30-min access to the diet, the bowls of diet were weighed. The difference of the weight from the beginning to the end of the test was the
The smallest dose of CCK-8 to decrease intake significantly compared to saline treatment was 2.0 mg/kg in rats on the 5% fat diet ( P < .01) and in rats on the 34% fat diet ( P < .05). The satiating potency of CCK-8 in rats ingesting a 34% fat diet was also not significantly different from the potency of CCK-8 in rats ingesting the 5% fat diet [ F(1,12) = 0.34, P=.57; Fig. 3]. There was a significant effect of dose of CCK-8 on intake of both diets [ F(5) = 7.76, P=.00], but there was no interaction between dose of CCK-8 and type of diet [ F(5,60) = 0.44, P=.82]. The body weight gained during the experiment by rats ingesting the 34% fat diet was not significantly different from the weight gained by rats ingesting the 5% fat ( P=.12; Table 2). There was also no significant effect of diet on the weight of the inguinal ( P=.07) and epididymal ( P=.87) fat pads (Table 2). 3.3. Discussion The threshold dose of CCK-8 was higher and equal on the two diets. This experiment failed to show the effect of the 34% fat diet on the sensitivity to CCK-8 that we observed in Experiment 1 and that Covasa and Ritter [1] reported. Thus, when we approximated their schedule of testing more closely, we did not get their result. We have no explanation for this.
Table 2 Fat pad weights and body weight gain on 5% and 34% fat diets
Epididymal fat pad Inguinal fat pad Body weight gain
5% fat
34% fat
8.1 ± 0.41 3.9 ± 0.71 279.4 ± 14.54
8.0 ± 0.87 5.8 ± 0.49 245.4 ± 13.43
Data are mean ± S.E. grams from five rats on a 5% fat diet and nine rats on a 34% fat diet. Body weight gain was the difference between body weights at the end and beginning of the experiment that lasted 13 weeks. There was no statistically significant effect of diet.
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
In contrast to the failure to replicate the effect of diet on threshold dose, this schedule of testing did replicate the lack of effect of the percentage of fat in the diet on the satiating potency of all of the doses of CCK-8 that we observed in the first experiment. The results of this experiment confirmed the lack of a significant difference in body weight gained by rats on the two diets observed in Experiment 1 (Table 2). The lack of effect of diet on the weight of epididymal and inguinal fat pads confirmed the results of Covasa et al. [7] who measured the epididymal, retroperitoneal, and subcutaneous fat obtained from rats maintained on 6% and 30.4% fat diets. They also reported that total carcass lipid was not significantly different on the two diets. All of these results are evidence that the diets did not have a differential effect on adiposity.
4. Experiment 3 This experiment differed from the first two by substituting a 60% fat diet for the 34% fat diet. The rationale of this experiment was to investigate whether a more concentrated fat diet that increased body weight and adiposity would produce a larger decrease in the satiating potency of CCK-8 than we observed in Experiment 1 (Fig. 4).
23
4.1. Methods The rats and the protocol were the same as were used in Experiment 1 except that the rats that ingested the 34% fat diet in Experiment 1 were maintained and tested on a 60% fat diet in this experiment (Table 1). The rats that ingested the 5% fat diet in Experiment 1 were tested on the same diet in this experiment. At the beginning of this experiment, rats on the 60% fat diet weighed 491.3 ± 15.6 g and the rats on the 5% fat diet weighed 480.7 ± 11.1 g. Rats were food deprived at 1630 h the night before testing. Five minutes before presentation of the diets, rats received an intraperitoneal injection (1 ml/kg) of either CCK-8 (1.0, 0.5, 0.25, and 0.125 mg/kg in that order) in saline or saline alone. Food bowls were refilled with fresh diets, weighed, and presented to the rats at 0930 h. After access to the diets for 30 min, food bowls were removed and weighed. Body weights were recorded daily just prior to the test. At the conclusion of the experiment, rats were sacrificed: epididymal and inguinal fat pads were dissected out of the rats, blotted with filter paper, and weighed. 4.1.1. Statistics The intakes after saline in rats on both diets could not be pooled because they were significantly different across test days ( P=.05). Therefore, we determined the threshold dose for significant inhibition by comparing the intake after a dose of CCK-8 to the intake after saline on the day that preceded the CCK test using paired t-test, 1-tailed. To determine the potency of CCK-8 in the rats on the two diets, we subtracted the intake after each dose of CCK-8 from the intake after saline on the preceding test day. These differences were then analyzed by two-way analysis of variance with repeated measures. To compare the results of the three experiments with the same metric, we reanalyzed the results with CCK-8 in Experiments 1 and 2 using differences. 4.2. Results
Fig. 4. Data are mean ± S.E. grams of diet eaten in 30 min in Experiment 3. Six rats were maintained on a 5% fat diet and six rats were maintained on a 60% fat diet. Diagonal columns are intakes of 5% fat diet after saline; empty columns are intakes of 5% fat diet after CCK-8; cross-hatched columns are intakes of 60% fat diet after saline; and black columns are intakes of 60% fat diet after CCK-8. * Significantly smaller intake than after saline, P < .05. y Significantly larger decrease of intake after the same dose of CCK-8 in rats maintained on a 60% fat diet than in rats maintained on a 5% fat diet, P < .05.
The threshold dose for decreased intake after CCK-8 was 0.125 mg/kg in rats on the 60% fat diet ( P < .01) and was 0.25 mg/kg ( P < .02) in the rats on the 5% fat diet. The 60% fat diet not only increased the sensitivity of the rats to CCK-8, it also significantly increased the potency of CCK-8 compared to its potency in rats on the 5% fat diet [ F(1,10) = 20.81, P < .01]. There was also a statistically significant effect of dose [ F(3) = 4.04, P=.02]. Although the interaction between dose and diet was not quite statistically significant [ F(3,30) = 2.86, P=.053], rats on the 60% fat diet inhibited intake significantly more after 0.25 ( P < .01) and 1.0 mg/kg ( P=.03) than rats on the 5% fat diet. The significant increase in potency of CCK-8 in rats on the 60% fat diet was not due to a significant difference in intakes of 60% fat (mean = 9.6 ± 1.1 g) or of 5% fat (mean = 7.9 ± 1.8 g) diets after saline injections [ F(1,10) = 3.76, P=.08].
24
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
Table 3 Fat pad weights and body weight gain in rats on 5% and 60% fat diets
Epididymal fat pad Inguinal fat pad Body weight gain
5% fat
60% fat
11.0 ± 0.99 6.7 ± 0.59 139.3 ± 12.42
18.1 ± 1.68 * 12.7 ± 1.29 * 207.8 ± 9.31 *
Data are mean ± S.E. grams from six rats on a 5% fat diet and six rats on a 60% fat diet. Body weight gain was the difference between body weights at the end and beginning of the experiment that lasted 7 weeks. * Significantly more than in rats on 5% diet, P .1.
Rats on the 60% fat diet gained significantly more body weight ( P < .01) and had heavier epididymal ( P < .01) and inguinal ( P < .01) fat pads than rats on the 5% fat diet (Table 3). 4.3. Discussion The increased satiating potency of CCK-8 in rats ingesting the 60% fat diet was unexpected. To our knowledge, this is the first report of increased satiating potency of CCK-8 correlated with chronic ingestion of a high-fat diet. The increased potency was characterized by the lowest threshold dose observed in the three experiments and by a significant increase in the overall inhibition of intake. The increased potency was measured when the differences between 30-min intakes after saline and the four doses of CCK-8 were used for statistical analysis. To determine whether this analysis biased the results of Experiment 3, we reanalyzed the results of the other two experiments as the difference between intakes after saline and CCK-8 after each dose of CCK-8. This analysis confirmed that the potency of CCK-8 in rats ingesting the 34% fat diet was not significantly different from rats ingesting the 5% fat diet [Experiment 1: F(1,10) = 1.19, P=.30; Experiment 2: F(1,12) = 0.22, P=.65]. We also reanalyzed the results of Experiments 1 and 3 because the same rats were used in both experiments. CCK-8 was significantly more potent in the same rats when they ingested the 60% diet in Experiment 3 than when they ingested the 34% diet in Experiment 1 [ F(1,10) = 8.05, P=.02]. In contrast, the potency of CCK-8 was not significantly different in the rats ingesting the 5% fat diet in Experiment 3 than when the same rats ingested the 5% fat diet in Experiment 1 [ F(1,10) = 2.33, P=.16]. Thus, the increased satiating potency of CCK-8 in rats ingesting the 60% fat diet and the lack of effect of the 34% fat diet were obtained when the data were analyzed in the same way and the same measure of potency was used. Enterostatin is the only other peripheral peptide that has increased inhibitory potency on food intake when it is administered to rats on a high-fat diet (30% or more) compared to rats on a low-fat diet [9,10]. It will be interesting to investigate whether the mechanisms that mediate the increased potency of enterostatin are related to those that mediate the increased potency of CCK-8 observed in rats on a 60% fat diet.
5. General discussion High-fat diets produced two effects on the sensitivity and potency of CCK-8. In the first experiment, the 34% fat diet increased the threshold dose of CCK-8 for inhibiting 30-min intake compared to the threshold dose in rats on a 5% fat diet. This change in sensitivity occurred without a significant change in the potency of the dose – response function. This confirmed the report of Covasa and Ritter [1]. When we attempted to replicate this result in Experiment 2, however, we failed. The reason for the failure to replicate is not clear. We followed detailed directions from Covasa and Ritter (from their papers and personal communications) for the preparation, storage, and presentation of the diets and used their protocol. Both laboratories have extensive experience with the use of CCK-8 so that any difference in the handling of CCK-8 is likely to be trivial. That leaves some subtle difference in experimental conditions as the most likely explanation, but we have not been able to identify anything beyond the fact that the potency of low doses of CCK-8 under our conditions appeared to be less than under theirs and that we obtained rats from a different supplier. Thus, the specific experimental conditions necessary for the 34% fat diet to reduce the sensitivity of rats to exogenous CCK-8 remain to be identified. In contrast to the effect of the 34% fat diet in the first experiment, when rats were maintained on a 60% fat diet in Experiment 3, the threshold dose of CCK-8 was lower and the satiating potency of CCK-8 was greater than in rats maintained on a 5% fat diet. Covasa and Ritter [1] did not observe this when they maintained rats on a 54% fat diet. It is not clear how the chronic ingestion of the 60% fat diet produced the increased potency of CCK-8. It is possible that duration of ingestion of a high-fat diet rather than percent of dietary fat was the relevant correlate of the unexpected increased satiating potency of CCK-8 because the rats that were maintained on 60% fat in Experiment 3 had been maintained on 34% fat prior to being switched to 60% fat. It is also possible that the effect of the 60% fat diet on the potency of CCK-8 was due to the increased body weight gain and adiposity because increased adiposity is correlated with increased plasma leptin and insulin [11,12]. Increased plasma leptin and insulin could increase the potency of CCK-8 in rats on a 60% fat diet because injections of leptin or insulin into the third cerebral ventricle increase the potency of exogenous CCK-8 IP [11,12]. Given the novelty of the result with the 60% fat diet and its potential importance for understanding the role of endogenous CCK in dietary-induced obesity, further experiments are required to determine the reproducibility and mechanisms of this effect.
Acknowledgements We thank Ms. Laurel Torres for assistance in processing the manuscript, and Drs. Nori Geary, James Gibbs, and Gary
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
Schwartz for constructive criticism of the penultimate draft. This research was supported by research grant MH 40010 from the National Institutes of Health. References [1] Covasa M, Ritter RC. Rats maintained on high-fat diets exhibit reduced satiety in response to CCK and bombesin. Peptides 1998;19(8): 1407 – 15. [2] Covasa M, Ritter RC. Adaptation to high-fat diet reduces inhibition of gastric emptying by CCK and intestinal oleate. Am J Physiol, Regul Integr Comp Physiol 2000;278(1):R166 – 70. [3] Covasa M, Grahn J, Ritter RC. High fat maintenance diet attenuates hindbrain neuronal response to CCK. Regul Pept 2000;86(1 – 3):83 – 8. [4] Brenner L, Yox DP, Ritter RC. Suppression of sham feeding by intraintestinal nutrients is not correlated with plasma cholecystokinin elevation. Am J Physiol 1993;264:R972 – 6. [5] Covasa M, Ritter RC. Reduced sensitivity to the satiation effect of intestinal oleate in rats adapted to high-fat diet. Am J Physiol 1999; 277(1 Pt 2):R279 – 85.
25
[6] Covasa M, Grahn J, Ritter RC. Reduced hindbrain and enteric neuronal response to intestinal oleate in rats maintained on high-fat diet. Auton Neurosci 2000;84(1 – 2):8 – 18. [7] Covasa M, Marcuson JK, Ritter RC. Diminished satiation in rats exposed to elevated levels of endogenous or exogenous cholecystokinin. Am J Physiol, Regul Integr Comp Physiol 2001;280(2):R331 – 7. [8] Smith GP. Cholecystokinin—the first twenty-five years. In: Bray GA, Ryan DH, editors. Nutrition, genetics, and obesity. Pennington Center Nutrition Series, vol. 9. Baton Rouge: Louisiana State Univ Press; 1999. p. 227 – 45. [9] York DA, Lin L, Smith BK, Bray GA. Enterostatin and 5HT: modulation of fat intake. In: Bray GA, Ryan DH, editors. Nutrition, genetics, and obesity. Pennington Center Nutrition Series, vol. 9. Baton Rouge: Louisiana State Univ Press; 1999. p. 246 – 66. [10] Lin L, York DA. Chronic ingestion of dietary fat is a prerequisite for inhibition of feeding by enterostatin. Am J Physiol 1998;275:619 – 23. [11] Woods SC, Seeley RJ, Porte Jr D, Schwartz MW. Signals that regulate food intake and energy homeostasis. Science 1998;280:1378 – 83. [12] Baskin DG, Figlewicz LD, Seeley RJ, Woods SC, Porte Jr D, Schwartz MW. Insulin and leptin: dual adiposity signals to the brain for the regulation of food intake and body weight. Brain Res 1999; 848(1 – 2):114 – 23.
Two effects of high-fat diets on the satiating potency of cholecystokinin-8 Ann-Marie Torregrossa, Gerard P. Smith* E.W. Bourne Laboratory, Department of Psychiatry, Weill Medical College of Cornell University, New York, NY, USA E.W. Bourne Laboratory, New York-Presbyterian Hospital, Westchester Division, 21 Bloomingdale Road, White Plains, NY 10605, USA Received 6 February 2002; received in revised form 1 July 2002; accepted 6 August 2002
Abstract Chronic ingestion of diets containing 34% or 54% fat have been reported [Peptides 19 (1998) 1407] to decrease the inhibitory effect on food intake of doses of cholecystokinin-8 (CCK-8) less than 1 mg/kg ip. We attempted to replicate this phenomenon in three experiments by comparing the effect of high-fat and low-fat diets on the threshold dose of CCK-8 for inhibition and on the dose – response function for doses of CCK-8 that ranged from 0.125 to 2.0 mg/kg. The first experiment tested rats five times per week. Rats on a 34% fat diet had a higher threshold (1.0 mg/kg) than rats on a 5% fat diet (0.25 mg/kg). The dose – response functions, however, were not significantly different. This result replicated the earlier report [Peptides 19 (1998) 1407]. The second experiment tested rats maintained on the same diets every other day as in the original report. It failed, however, to replicate the results of the first experiment or the earlier report because the threshold doses and the dose – response functions of CCK-8 were not significantly different between rats on 34% and 5% fat diets. The third experiment compared the potency of CCK-8 in rats on a 60% fat diet with the potency in rats on a 5% fat diet. CCK-8 was significantly more potent in the rats on the 60% fat diet because the threshold dose of these rats was lower (0.125 mg/kg) than in rats on the 5% fat diet (0.25 mg/kg), and the dose – response function in rats on the 60% fat diet was significantly more potent than in rats on the 5% fat diet. Thus, we observed two effects of the chronic ingestion of high-fat diets on the inhibitory potency of CCK-8: (1) A 34% fat diet increased the threshold dose of CCK-8 without changing the dose – response function in one of two experiments. (2) A 60% fat diet increased the potency of CCK-8 because it decreased the threshold dose and increased the dose – response function significantly. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Control of food intake; Gut peptides; Cholecystokinin-33; Diet-induced obesity; Fat pads; Satiation
1. Introduction In 1998, Covasa and Ritter [1] reported that low doses of cholecystokinin-8 (CCK-8) administered intraperitoneally decreased 30-min intake significantly less in rats maintained on a 34% fat diet than in rats maintained on a 5% fat diet. The 34% fat diet decreased the potency of doses of CCK-8 smaller than 1 mg/kg, but it had no effect on the inhibitory potency of 1 mg/kg CCK-8. Thus, the chronic ingestion of the 34% fat diet decreased the sensitivity of rats to the satiating potency of CCK-8, but it apparently did not shift the upper part of the dose – response function to the right. These experiments also demonstrated that the effect of the 34% fat diet depended on rats eating the diet for at least 2 weeks prior to the intake tests. The authors concluded that the effect of the 34% fat diet was not due to the fat content of * Corresponding author. E.W. Bourne Laboratory, New York-Presbyterian Hospital, Westchester Division, 21 Bloomingdale Road, White Plains, NY 10605, USA. Tel.: +1-914-997-5935; fax: +1-914-682-3793. E-mail address: [email protected] (G.P. Smith).
the diet that rats ate during the intake test, to the caloric density of the diet, or to a difference in the amount of body weight gained on the 5% and 34% diets. This interesting effect of a high-fat diet on the potency of low doses of exogenous CCK-8 was followed up in two experiments. First, CCK-8 (0.25 –2.0 mg/kg ip) decreased gastric emptying less in rats on a 54% fat diet than in rats on a 5% fat diet [2]. Second, CCK-8 (0.25 mg/kg ip) produced a significantly smaller number of cells with Fos-like immunoreactivity (Fos LI) in the dorsal vagal complex in rats on a 34% fat diet than in rats on a 5% fat diet [3]. These results with exogenous CCK-8 were apparently extended to endogenous CCK by using intestinal infusions of oleate that had been shown to decrease food intake by releasing endogenous CCK [4]. In comparison to low-fat diets, high-fat diets (34% or 54%) decreased the inhibitory effect of duodenal infusions of oleate on intake [5], gastric emptying [2], and the number of Fos LI cells in the dorsal vagal complex [6]. The parallel results of high-fat diets on the inhibitory potencies of exogenous CCK-8 and intraintestinal oleate
0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 8 8 8 - 0
20
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
were consistent with the possibility that the decreased response to oleate was due to the decreased response to endogenous CCK released by oleate [4]. No direct evidence, however, was presented for this causal relationship. In a recent experiment, Covasa et al. [7] demonstrated that when circulating CCK was increased chronically by continuous intraperitoneal infusion for 28 days, the potency of an intraperitoneal injection of CCK-8 was reduced. This result suggested that chronic elevation of CCK produced by chronic ingestion of a high-fat diet could be an important cause for the reduced potency of CCK-8 observed with that diet. The mechanism of the reduced response to CCK-8 or intestinal oleate is unknown. The mechanism could be peripheral, involving a reduction in receptor binding due to down-regulation of the number of receptors or a reduced transduction by vagal afferent neurons that mediate the satiating effect of CCK-8 and intraintestinal oleate. On the other hand, the mechanism could be central—the change in metabolic state produced by the chronic consumption of a high-fat diet could alter the central processing of the peripheral vagal afferent information. Given our long-standing interest in the satiating effect of exogenous and endogenous CCK [8], we decided to investigate the site and kind of mechanism underlying the effect of high-fat diets. We began by attempting to reproduce the original report of the effect of a 34% diet on the potency of CCK-8.
2. Experiment 1 2.1. Methods 2.1.1. General conditions Twelve male Sprague Dawley rats (Taconic Farms, Germantown, NY), weighing 337– 402 g at the beginning of the experiment, were housed individually in polycarbonate, solid-bottom cages (24 20 29 cm) with cob bedding. The lighting schedule was a 12-h, light/dark cycle; lights were on from 0700 to 1900 h. All rats had access to tap water ad libitum. Six rats (372.3 ± 9.5 g) were maintained on a 34% fat diet and 6 (373.5 ± 8.3 g) were maintained on a 5% fat diet. The preparation and constituents of the diets (Table 1) were identical to the diets used by Covasa and Ritter [1]. Note that the 5% fat diet is high in carbohydrate in the form of corn starch and that the 34% fat diets contain no carbohydrate. Diets were presented in spill-resistant metal bowls (Lab. Products, Seaford, DE) resting in larger ceramic bowls to collect any spillage. Rats were adapted to their assigned diet for at least 2 weeks prior to testing. 2.1.2. Test schedule Rats were tested daily (Monday – Friday). Diets were removed at 1630 h and weighed bowls of fresh diet were returned at 0930 h. Five minutes before the presentation of the diets, all rats received an intraperitoneal injection (1 ml/
Table 1 Diet constituents (wt.%)
5% fat
34% fat
60% fat
Casein Corn starch Fat Vitamin mix Mineral mix Alphacel DL-Methionine
20 65.4 5 2.2 4 3 0.4
20 0 34 2.2 4 39.4 0.4
20 0 60 2.2 4 13.4 0.4
The composition of the 5% and 34% fat diets are from Ref. [1]. The 60% fat diet was used in Experiment 3. The preparation of the diets was as described in Ref. [1] and in personal communication with Covasa and Ritter. Casein and methionine were purchased from Sigma (St. Louis, MO). Alphacel and corn starch were purchased from ICN Biomedicals (Aurora, OH). The fat mixture consisted of 1% Canola vegetable oil (Pure Wesson Canola Oil, Fullerton, CA) and 4% Lard (Armour Lard, Omaha, NE) and it was the one used by Covasa and Ritter (personal communication). The vitamin and mineral mixes were purchased from Bio-Serve (Frenchtown, NJ).
kg) of either CCK-8 (Peninsula Laboratories, Belmont, CA; 1.0, 0.5, 0.25, 0.125, and 2.0 mg/kg in that order) in 0.9% saline, equimolar doses of porcine CCK-33 (Peptides International, Louisville, KY; 3.3, 1.65, 0.83, 0.42, and 6.6 mg/kg in that order) in 0.9% of saline, or 0.9% saline (bacteriostatic 0.9% sodium chloride, Abbott Laboratories, North Chicago, IL). Each dose of both peptides was tested once. The tests with CCK-8 were completed before the tests with CCK-33 began. At least one test after an injection of 0.9% saline preceded a test in which CCK-8 or CCK-33 was injected. After 30-min access to the diet, the bowls of diet were weighed again and the difference in weight from the beginning of the test was the measure of intake because spillage did not occur during the tests. Body weights were recorded prior to each test. After the test on Friday, rats had access to their diets ad libitum until 1630 h on Sunday. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Weill Medical College of Cornell University. 2.1.3. Statistics Separate two-way analysis of variance with repeated measures on dose of peptide were used to analyze the effect of the two fat diets on the satiating potency of all of the doses of CCK-8 and CCK-33. We used planned comparisons by ttests to determine the smallest dose of CCK-8 or CCK-33 that inhibited intake compared to the mean intake after saline treatment in Experiments 1 and 2. Because the intakes after saline in Experiment 3 differed significantly over time ( P=.05), the intake after CCK-8 was compared to the intake after saline on the day that preceded that dose in that experiment. Given the extensive evidence that CCK-8 inhibits intake, but does not increase it, we considered that a difference between intakes after CCK-8 or CCK-33 and saline was significant when P=.05 or less, one-tailed. This estimate of the threshold dose for inhibition is a measure of the sensitivity that was reported to be changed by the high-fat diet [1].
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
21
The effects of fat diets on body weight gain were analyzed with t-tests, two-tailed. Differences were considered significant when P=.05 or less. All statistical analyses were performed using SYSTAT 7.0 statistical analysis software program and graphics were created with Sigma Plot for Windows 2.0 and 3.0. 2.2. Results The smallest dose of CCK-8 that inhibited intake significantly was 0.25 mg/kg in rats on the 5% fat diet ( P < .03) and 1 mg/kg in rats on the 34% fat diet ( P=.00). The smallest dose of CCK-33 that inhibited intake significantly was 0.83 mg/kg in rats on the 5% fat diet ( P=.02) and 1.65 mg/kg in rats on the 34% fat diet ( P < .01). Note that the equimolar threshold doses of CCK-33 and CCK-8 were the same on the 5% fat diet, but the threshold dose CCK-33 was smaller than that of CCK-8 in rats on the 34% fat diet. In contrast, the percentage of fat in the diet did not produce a significant difference in the satiating potency of CCK-8 [ F(1,10) = 2.28, P=.16; Fig. 1] or CCK-33 [ F(1,10) = 0.37, P=.56; Fig. 2]. There was, however, a significant effect of dose of CCK-8 [ F(5) = 17.76, P=.00] and dose of CCK-33 [ F(5)=37.75, P=.00] on the intakes of both diets, but no interaction between diet and dose of CCK-8 [ F(5,50) = 0.85, P=.52] or CCK-33 [ F(5,50) = 1.56, P=.19]. The diets did not produce a significant difference in weight gain from the beginning to the end of the experiment (5%, 105.7 ± 11.27 g; 34%, 93.7 ± 3.69 g; P=.35).
Fig. 2. Data are mean ± S.E. grams of diet eaten in 30 min. Empty columns are intakes of 5% fat diet (n = 6); black columns are intakes of 34% fat diet (n = 6). Doses of CCK-33 were equimolar to the doses of CCK-8 in Fig. 1. Intakes after the zero dose are the pooled intakes from the five tests after saline administration on the days that preceded CCK-33 tests. * Smallest dose of CCK-33 that produced a significantly smaller intake than the intake after saline, P < .05.
2.3. Discussion The threshold dose in rats on the 34% fat diet was four times as large for CCK-8 and twice as large for CCK-33 as the threshold dose in rats on the 5% fat diet. Thus, rats on the 34% fat diet were less sensitive to the satiating effect of CCK-8 and CCK-33 than rats on the 5% fat diet. The satiating potencies of all of the doses of CCK-8 and CCK-33 in rats ingesting the 34% fat diet, however, were not significantly different from their satiating potencies in rats ingesting the 5% fat diet. This combination of decreased sensitivity for low doses of CCK-8 and no change in the dose – response function is consistent with the results of Covasa and Ritter [1]. The same combination of results with CCK-33 extends the effect of 34% fat diets to a larger form of CCK. There was a difference in the test schedule between our protocol and that of Covasa and Ritter [1]. We tested our animals daily, Monday through Friday, while Covasa and Ritter [1] tested theirs every other day. Because the difference in test frequency might influence the results, we repeated the experiment using their protocol.
Fig. 1. Data are mean ± S.E. grams of diet eaten in 30 min. Empty columns are intakes of 5% fat diet (n = 6); black columns are intakes of 34% fat diet (n = 6). Intakes after the zero dose are the pooled intakes from the five tests after saline administration on the days that preceded CCK-8 tests. * Smallest dose of CCK-8 that produced a significantly smaller intake than the intake after saline, P < .05.
3. Experiment 2 There were three differences between this experiment and Experiment 1. One was the schedule of testing. In Experiment 1, rats were tested daily; in this experiment, they were
22
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
measure of intake. After the test on Friday, rats had access to the diets ad libitum until 1630 h on Sunday. Body weights of all rats were recorded daily. This occurred prior to the test on test days or at the same time on Tuesdays and Thursdays. Rats were sacrificed at the conclusion of the experiment and epididymal and inguinal fat pads were dissected out of the animals, blotted with filter paper, and weighed. 3.1.1. Statistics The effect of the fat diets on threshold dose for inhibition of intake, on the potency of all of the doses of CCK-8, and on body weight gain were analyzed exactly as in Experiment 1. The effect of fat diets on the weights of inguinal and epididymal fat pads were analyzed with t-tests, two-tailed. Differences were considered significant when P=.05 or less. 3.2. Results Fig. 3. Data are mean ± S.E. grams of diet eaten in 30 min in Experiment 2. Empty columns are intakes of 5% fat diet (n = 5); black columns are intakes of 34% fat diet (n = 9). * Smallest dose of CCK-8 that produced a significantly smaller intake than the intake after saline, P < .05.
tested every other day. The second difference is that we tested only CCK-8 because the results with CCK-8 and CCK-33 were similar in Experiment 1. The third difference is that we measured the weight of inguinal and epididymal fat pads at the end of the experiment to determine if the 34% fat diet increased the weight of these fat pads more than 5% fat diet even though the 34% fat diet had not increased body weight in Experiment 1 more than the 5% fat diet did. 3.1. Methods Fourteen male Sprague Dawley Rats (Taconic Farms), weighing 172– 222 g at the beginning of the experiment, were used. These rats had no prior experience with the test diets or with the test protocol. All housing, lighting conditions, and diets were as described in Experiment 1. Nine rats (197.0 ± 5.1 g) were maintained on the 34% fat diet and five rats (203.4 ± 8.5 g) were maintained on the 5% fat diet. All rats had tap water ad libitum. Rats were adapted to their assigned diet for at least 2 weeks prior to testing. Diet was presented in spill-resistant metal bowls resting in larger ceramic bowls on top of plastic trays (M. Covasa, personal communication). Animals were tested on Mondays, Wednesdays, and Fridays after being deprived of their diet at 1630 h on the prior day. At 0930 h, the food bowls were filled with fresh diet, weighed, and presented. Five minutes before presentation of the diets, rats received an intraperitoneal injection (1 ml/kg) of either CCK-8 (1.0, 0.5, 0.25, 0.125, and 2.0 mg/kg in that order) in saline or saline alone. After 30-min access to the diet, the bowls of diet were weighed. The difference of the weight from the beginning to the end of the test was the
The smallest dose of CCK-8 to decrease intake significantly compared to saline treatment was 2.0 mg/kg in rats on the 5% fat diet ( P < .01) and in rats on the 34% fat diet ( P < .05). The satiating potency of CCK-8 in rats ingesting a 34% fat diet was also not significantly different from the potency of CCK-8 in rats ingesting the 5% fat diet [ F(1,12) = 0.34, P=.57; Fig. 3]. There was a significant effect of dose of CCK-8 on intake of both diets [ F(5) = 7.76, P=.00], but there was no interaction between dose of CCK-8 and type of diet [ F(5,60) = 0.44, P=.82]. The body weight gained during the experiment by rats ingesting the 34% fat diet was not significantly different from the weight gained by rats ingesting the 5% fat ( P=.12; Table 2). There was also no significant effect of diet on the weight of the inguinal ( P=.07) and epididymal ( P=.87) fat pads (Table 2). 3.3. Discussion The threshold dose of CCK-8 was higher and equal on the two diets. This experiment failed to show the effect of the 34% fat diet on the sensitivity to CCK-8 that we observed in Experiment 1 and that Covasa and Ritter [1] reported. Thus, when we approximated their schedule of testing more closely, we did not get their result. We have no explanation for this.
Table 2 Fat pad weights and body weight gain on 5% and 34% fat diets
Epididymal fat pad Inguinal fat pad Body weight gain
5% fat
34% fat
8.1 ± 0.41 3.9 ± 0.71 279.4 ± 14.54
8.0 ± 0.87 5.8 ± 0.49 245.4 ± 13.43
Data are mean ± S.E. grams from five rats on a 5% fat diet and nine rats on a 34% fat diet. Body weight gain was the difference between body weights at the end and beginning of the experiment that lasted 13 weeks. There was no statistically significant effect of diet.
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
In contrast to the failure to replicate the effect of diet on threshold dose, this schedule of testing did replicate the lack of effect of the percentage of fat in the diet on the satiating potency of all of the doses of CCK-8 that we observed in the first experiment. The results of this experiment confirmed the lack of a significant difference in body weight gained by rats on the two diets observed in Experiment 1 (Table 2). The lack of effect of diet on the weight of epididymal and inguinal fat pads confirmed the results of Covasa et al. [7] who measured the epididymal, retroperitoneal, and subcutaneous fat obtained from rats maintained on 6% and 30.4% fat diets. They also reported that total carcass lipid was not significantly different on the two diets. All of these results are evidence that the diets did not have a differential effect on adiposity.
4. Experiment 3 This experiment differed from the first two by substituting a 60% fat diet for the 34% fat diet. The rationale of this experiment was to investigate whether a more concentrated fat diet that increased body weight and adiposity would produce a larger decrease in the satiating potency of CCK-8 than we observed in Experiment 1 (Fig. 4).
23
4.1. Methods The rats and the protocol were the same as were used in Experiment 1 except that the rats that ingested the 34% fat diet in Experiment 1 were maintained and tested on a 60% fat diet in this experiment (Table 1). The rats that ingested the 5% fat diet in Experiment 1 were tested on the same diet in this experiment. At the beginning of this experiment, rats on the 60% fat diet weighed 491.3 ± 15.6 g and the rats on the 5% fat diet weighed 480.7 ± 11.1 g. Rats were food deprived at 1630 h the night before testing. Five minutes before presentation of the diets, rats received an intraperitoneal injection (1 ml/kg) of either CCK-8 (1.0, 0.5, 0.25, and 0.125 mg/kg in that order) in saline or saline alone. Food bowls were refilled with fresh diets, weighed, and presented to the rats at 0930 h. After access to the diets for 30 min, food bowls were removed and weighed. Body weights were recorded daily just prior to the test. At the conclusion of the experiment, rats were sacrificed: epididymal and inguinal fat pads were dissected out of the rats, blotted with filter paper, and weighed. 4.1.1. Statistics The intakes after saline in rats on both diets could not be pooled because they were significantly different across test days ( P=.05). Therefore, we determined the threshold dose for significant inhibition by comparing the intake after a dose of CCK-8 to the intake after saline on the day that preceded the CCK test using paired t-test, 1-tailed. To determine the potency of CCK-8 in the rats on the two diets, we subtracted the intake after each dose of CCK-8 from the intake after saline on the preceding test day. These differences were then analyzed by two-way analysis of variance with repeated measures. To compare the results of the three experiments with the same metric, we reanalyzed the results with CCK-8 in Experiments 1 and 2 using differences. 4.2. Results
Fig. 4. Data are mean ± S.E. grams of diet eaten in 30 min in Experiment 3. Six rats were maintained on a 5% fat diet and six rats were maintained on a 60% fat diet. Diagonal columns are intakes of 5% fat diet after saline; empty columns are intakes of 5% fat diet after CCK-8; cross-hatched columns are intakes of 60% fat diet after saline; and black columns are intakes of 60% fat diet after CCK-8. * Significantly smaller intake than after saline, P < .05. y Significantly larger decrease of intake after the same dose of CCK-8 in rats maintained on a 60% fat diet than in rats maintained on a 5% fat diet, P < .05.
The threshold dose for decreased intake after CCK-8 was 0.125 mg/kg in rats on the 60% fat diet ( P < .01) and was 0.25 mg/kg ( P < .02) in the rats on the 5% fat diet. The 60% fat diet not only increased the sensitivity of the rats to CCK-8, it also significantly increased the potency of CCK-8 compared to its potency in rats on the 5% fat diet [ F(1,10) = 20.81, P < .01]. There was also a statistically significant effect of dose [ F(3) = 4.04, P=.02]. Although the interaction between dose and diet was not quite statistically significant [ F(3,30) = 2.86, P=.053], rats on the 60% fat diet inhibited intake significantly more after 0.25 ( P < .01) and 1.0 mg/kg ( P=.03) than rats on the 5% fat diet. The significant increase in potency of CCK-8 in rats on the 60% fat diet was not due to a significant difference in intakes of 60% fat (mean = 9.6 ± 1.1 g) or of 5% fat (mean = 7.9 ± 1.8 g) diets after saline injections [ F(1,10) = 3.76, P=.08].
24
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
Table 3 Fat pad weights and body weight gain in rats on 5% and 60% fat diets
Epididymal fat pad Inguinal fat pad Body weight gain
5% fat
60% fat
11.0 ± 0.99 6.7 ± 0.59 139.3 ± 12.42
18.1 ± 1.68 * 12.7 ± 1.29 * 207.8 ± 9.31 *
Data are mean ± S.E. grams from six rats on a 5% fat diet and six rats on a 60% fat diet. Body weight gain was the difference between body weights at the end and beginning of the experiment that lasted 7 weeks. * Significantly more than in rats on 5% diet, P .1.
Rats on the 60% fat diet gained significantly more body weight ( P < .01) and had heavier epididymal ( P < .01) and inguinal ( P < .01) fat pads than rats on the 5% fat diet (Table 3). 4.3. Discussion The increased satiating potency of CCK-8 in rats ingesting the 60% fat diet was unexpected. To our knowledge, this is the first report of increased satiating potency of CCK-8 correlated with chronic ingestion of a high-fat diet. The increased potency was characterized by the lowest threshold dose observed in the three experiments and by a significant increase in the overall inhibition of intake. The increased potency was measured when the differences between 30-min intakes after saline and the four doses of CCK-8 were used for statistical analysis. To determine whether this analysis biased the results of Experiment 3, we reanalyzed the results of the other two experiments as the difference between intakes after saline and CCK-8 after each dose of CCK-8. This analysis confirmed that the potency of CCK-8 in rats ingesting the 34% fat diet was not significantly different from rats ingesting the 5% fat diet [Experiment 1: F(1,10) = 1.19, P=.30; Experiment 2: F(1,12) = 0.22, P=.65]. We also reanalyzed the results of Experiments 1 and 3 because the same rats were used in both experiments. CCK-8 was significantly more potent in the same rats when they ingested the 60% diet in Experiment 3 than when they ingested the 34% diet in Experiment 1 [ F(1,10) = 8.05, P=.02]. In contrast, the potency of CCK-8 was not significantly different in the rats ingesting the 5% fat diet in Experiment 3 than when the same rats ingested the 5% fat diet in Experiment 1 [ F(1,10) = 2.33, P=.16]. Thus, the increased satiating potency of CCK-8 in rats ingesting the 60% fat diet and the lack of effect of the 34% fat diet were obtained when the data were analyzed in the same way and the same measure of potency was used. Enterostatin is the only other peripheral peptide that has increased inhibitory potency on food intake when it is administered to rats on a high-fat diet (30% or more) compared to rats on a low-fat diet [9,10]. It will be interesting to investigate whether the mechanisms that mediate the increased potency of enterostatin are related to those that mediate the increased potency of CCK-8 observed in rats on a 60% fat diet.
5. General discussion High-fat diets produced two effects on the sensitivity and potency of CCK-8. In the first experiment, the 34% fat diet increased the threshold dose of CCK-8 for inhibiting 30-min intake compared to the threshold dose in rats on a 5% fat diet. This change in sensitivity occurred without a significant change in the potency of the dose – response function. This confirmed the report of Covasa and Ritter [1]. When we attempted to replicate this result in Experiment 2, however, we failed. The reason for the failure to replicate is not clear. We followed detailed directions from Covasa and Ritter (from their papers and personal communications) for the preparation, storage, and presentation of the diets and used their protocol. Both laboratories have extensive experience with the use of CCK-8 so that any difference in the handling of CCK-8 is likely to be trivial. That leaves some subtle difference in experimental conditions as the most likely explanation, but we have not been able to identify anything beyond the fact that the potency of low doses of CCK-8 under our conditions appeared to be less than under theirs and that we obtained rats from a different supplier. Thus, the specific experimental conditions necessary for the 34% fat diet to reduce the sensitivity of rats to exogenous CCK-8 remain to be identified. In contrast to the effect of the 34% fat diet in the first experiment, when rats were maintained on a 60% fat diet in Experiment 3, the threshold dose of CCK-8 was lower and the satiating potency of CCK-8 was greater than in rats maintained on a 5% fat diet. Covasa and Ritter [1] did not observe this when they maintained rats on a 54% fat diet. It is not clear how the chronic ingestion of the 60% fat diet produced the increased potency of CCK-8. It is possible that duration of ingestion of a high-fat diet rather than percent of dietary fat was the relevant correlate of the unexpected increased satiating potency of CCK-8 because the rats that were maintained on 60% fat in Experiment 3 had been maintained on 34% fat prior to being switched to 60% fat. It is also possible that the effect of the 60% fat diet on the potency of CCK-8 was due to the increased body weight gain and adiposity because increased adiposity is correlated with increased plasma leptin and insulin [11,12]. Increased plasma leptin and insulin could increase the potency of CCK-8 in rats on a 60% fat diet because injections of leptin or insulin into the third cerebral ventricle increase the potency of exogenous CCK-8 IP [11,12]. Given the novelty of the result with the 60% fat diet and its potential importance for understanding the role of endogenous CCK in dietary-induced obesity, further experiments are required to determine the reproducibility and mechanisms of this effect.
Acknowledgements We thank Ms. Laurel Torres for assistance in processing the manuscript, and Drs. Nori Geary, James Gibbs, and Gary
A.-M. Torregrossa, G.P. Smith / Physiology & Behavior 78 (2003) 19–25
Schwartz for constructive criticism of the penultimate draft. This research was supported by research grant MH 40010 from the National Institutes of Health. References [1] Covasa M, Ritter RC. Rats maintained on high-fat diets exhibit reduced satiety in response to CCK and bombesin. Peptides 1998;19(8): 1407 – 15. [2] Covasa M, Ritter RC. Adaptation to high-fat diet reduces inhibition of gastric emptying by CCK and intestinal oleate. Am J Physiol, Regul Integr Comp Physiol 2000;278(1):R166 – 70. [3] Covasa M, Grahn J, Ritter RC. High fat maintenance diet attenuates hindbrain neuronal response to CCK. Regul Pept 2000;86(1 – 3):83 – 8. [4] Brenner L, Yox DP, Ritter RC. Suppression of sham feeding by intraintestinal nutrients is not correlated with plasma cholecystokinin elevation. Am J Physiol 1993;264:R972 – 6. [5] Covasa M, Ritter RC. Reduced sensitivity to the satiation effect of intestinal oleate in rats adapted to high-fat diet. Am J Physiol 1999; 277(1 Pt 2):R279 – 85.
25
[6] Covasa M, Grahn J, Ritter RC. Reduced hindbrain and enteric neuronal response to intestinal oleate in rats maintained on high-fat diet. Auton Neurosci 2000;84(1 – 2):8 – 18. [7] Covasa M, Marcuson JK, Ritter RC. Diminished satiation in rats exposed to elevated levels of endogenous or exogenous cholecystokinin. Am J Physiol, Regul Integr Comp Physiol 2001;280(2):R331 – 7. [8] Smith GP. Cholecystokinin—the first twenty-five years. In: Bray GA, Ryan DH, editors. Nutrition, genetics, and obesity. Pennington Center Nutrition Series, vol. 9. Baton Rouge: Louisiana State Univ Press; 1999. p. 227 – 45. [9] York DA, Lin L, Smith BK, Bray GA. Enterostatin and 5HT: modulation of fat intake. In: Bray GA, Ryan DH, editors. Nutrition, genetics, and obesity. Pennington Center Nutrition Series, vol. 9. Baton Rouge: Louisiana State Univ Press; 1999. p. 246 – 66. [10] Lin L, York DA. Chronic ingestion of dietary fat is a prerequisite for inhibition of feeding by enterostatin. Am J Physiol 1998;275:619 – 23. [11] Woods SC, Seeley RJ, Porte Jr D, Schwartz MW. Signals that regulate food intake and energy homeostasis. Science 1998;280:1378 – 83. [12] Baskin DG, Figlewicz LD, Seeley RJ, Woods SC, Porte Jr D, Schwartz MW. Insulin and leptin: dual adiposity signals to the brain for the regulation of food intake and body weight. Brain Res 1999; 848(1 – 2):114 – 23.