Effects of oral preload, CCK or bombesin administration on short term food intake of melanocortin 4-receptor knockout (MC4RKO) mice

peptides 27 (2006) 3226–3233

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Effects of oral preload, CCK or bombesin administration on short term food intake of melanocortin 4-receptor knockout (MC4RKO) mice C.H. Vaughan a,*, C. Haskell-Luevano b, A. Andreasen b, N.E. Rowland a a b

Department of Psychology, University of Florida, Gainesville, FL 32611, United States Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610, United States

article info

abstract

Article history:

We investigated whether either heterozygous (HET) or homozygous (knockout, KO) disrup-

Received 8 June 2006

tion of the melanocortin type 4 receptor (MC4R) gene alters post ingestive responsiveness of

Received in revised form

mice. Specifically, we tested the hypothesis that hyperphagia in MC4RKO mice might be due

30 July 2006

to a deficit in processes that sustain intermeal intervals (satiety) and/or processes that

Accepted 3 August 2006

terminate ongoing episodes of eating (satiation). To test satiety, mice drank an oral preload

Published on line 11 September 2006

and then we monitored intake of a subsequent liquid diet test meal. To test satiation, we examined the effect of exogenous administration of cholecystokinin (CCK) and bombesin

Keywords:

(BN) on the size of a liquid diet meal. Experiment 1 was comprised of two studies. In the first,

Cholecystokinin

we determined that the intake of all three genotypes following fasts of either 6, 12, or 24 h

Bombesin

were comparable, and so chose 12 h deprivation for the subsequent studies. In the second,

MC4RKO mice

12 h fasted mice were allowed to consume a fixed preload, approximately 50% of their

Preload

expected mean intake and, following delays of either 30 or 60 min, were allowed to consume

Gut

to satiation. Compared with no preload, the preload significantly reduced meal size com-

Satiety

parably in all three genotypes. The reduction in intake was greater when the test meal was presented 30 compared with 60 min after the preload, again with no genotype differences in

Abbreviations:

this decay of satiety. In experiment 2, we administered either CCK or BN and examined

CCK, cholecystokinin

suppression of meal size after a 12 h fast. Mice were tested repeatedly with CCK-8 (2, 6, or

BN, bombesin

18 mg/kg ip) or BN (2, 4 or 8 mg/kg ip) with vehicle injection days intervening. The 30 min

MC4RKO, melanocortin 4 receptor

intakes of HET and KO mice were suppressed more than those of WT following either CCK or

knockout

BN. These experiments suggest that diminished responsiveness to nutrients or gut satiety

WT, wild type

hormones is not responsible for hyperphagia in MC4RKO mice.

HET, heterozygous NTS, nucleus of the solitary tract NMB, neuromedin B GRP, gastrin releasing peptide ip, intraperitoneal

* Corresponding author. E-mail address: [email protected] (C.H. Vaughan). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.08.002

# 2006 Elsevier Inc. All rights reserved.

peptides 27 (2006) 3226–3233

Changes in meal frequency are usually attributed to modulation of processes that sustain intermeal intervals (satiety) whereas changes in meal size are attributed to modulation in the processes that terminate ongoing episodes of eating (satiation). The study of single meals has been used extensively to assess how endogenous and exogenous agents affect both satiation and satiety. Melanocortin receptor 4 knockout (MC4RKO) mice exhibit robust hyperphagia [22] but there have been relatively few investigations of their satiety signaling after individual meals. The fact that they are spontaneously hyperphagic suggests that MC4RKO mice are deficient in regulating meal size and/or frequency based on food already consumed [23,48,56]. The relationship between the MC4R and post ingestive feedback has been investigated both pharmacologically [4] and in MC4RKO mice [5] and suggest that impaired MC4R signaling impairs detection of changes in gastric volume. To further this line of inquiry we conducted two experiments to assess, directly and indirectly, the sensitivity of MC4RKO mice to gastric and postgastric feedback signals relating to meal termination. In our first experiment, we examined whether MC4RKO and wild type (WT) mice show comparable responses to graded levels of food deprivation, which is often used to generate predictable intake after a preload [41,44,45,61]. This was done because MC4RKO and WT mice may have different metabolic rates [1,10] and a given period of deprivation may have different behavioral effects on these genotypes. We then determined whether the satiating power of an oral preload would differ between KO, heterozygous (HET) and WT mice. In our second experiment, we tested short term intake after exogenous administration of cholecystokinin (CCK) or bombesin (BN). CCK, an endogenous octapeptide, is a putative satiety hormone that is secreted in response to ingestion of food, and is found in brain and gut [28,43,47]. CCK binds to two classes of receptors, CCKA and CCKB both of which are found peripherally and centrally [21,46]. Meal termination effects of CCK are mediated through CCKA receptors on vagal afferent fibers [13,18,38,62,63]. The vagus nerve relays the visceral information to the brainstem that in turn sends signals to various hypothalamic nuclei involved in inhibiting food intake (reviewed in Ref. [7]). The results of studies using selectively bred rodents [30] or pharmacological agents [64], as well as neuroanatomical evidence [16,39,52], suggest that expression of both CCKA and MC4 receptors in the NTS are involved in control of meal size. BN and its mammalian homologs neuromedin B (NMB) and gastrin releasing peptide (GRP) [33] decrease food intake by decreasing meal size and increasing the intermeal interval [2,17,19,20,51,54,65]. Normally, GRP is released prandially and increased levels in the hypothalamus are associated with the cessation of food consumption [37,42]. Endogenous GRP and exogenous BN bind to BB2 (GRP) receptors resulting in behavioral effects as well as CCKAR activation [24,26,29,47,65]. We tested both of these satiety signals for two purposes. First, MC4RKO mice may show differential sensitivity to one but not the other, a dissociation shown previously in rats [34]. Second, the site of action of each peptide is different. The satiety effect of peripheral BN requires both vagal and splanchnic visceral input whereas CCK requires gastric branches of the vagus [20]. One study to date [16] has

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examined the response of MC4RKO mice to CCK-8 (3 nmol/ kg) and reported that the peptide has no anorectic effect in MC4RKO mice compared with WT mice. We now examine the effects of CCK and BN using a range of doses. If the hyperphagia of MC4RKO mice is due to defective signaling from either of these endogenous prandially released hormones, then we hypothesize that the dose–response curve of either or both will be shifted to the right in the KO mice in comparison to WT mice.

1.

Materials and methods

1.1.

Animals

The vivarium was illuminated from 07:00 to 19:00 h and was maintained at an ambient temperature of 23  2 8C. Mice were housed individually in standard shoebox cages with water and food (Purina 5001 Chow; 3.6 kcal/g) available ad libitum, unless otherwise noted. All mice were obtained from the breeding colony of Dr. Haskell-Luevano at the University of Florida. Animal use in this study was approved by the University of Florida IACUC.

1.2. Experiment 1: effect of food deprivation then preload on test meal intake Sixty mice (20 WT, 20 HET, 20 KO) were used in this experiment. Fifty were born in the Psychology Department from mothers donated by Dr. Haskell-Luevano and were used in previous experiments. Six of the 50 aforementioned mice were used in a previous experiment where mice had to lever press for food in an operant chamber. Ten additional mice were donated as adults by Dr. Haskell-Luevano and were experimentally naı¨ve. Ages of mice ranged from 4 to 12 months old. We noticed no systematic differences in the results between the mice from these different origins. For fasting and preload measurements, the spacing of testing days was partially dependent on time constraints of fasting and weight maintenance of the mice.

1.2.1.

Food deprivation study

All mice were adapted to consume Ensure1 (Ross Laboratories, Columbus, OH; 14.4% protein, 64% carbohydrate, 21.6% fat) by attaching 10 ml pipettes, fitted with metal drinking spouts and rubber stoppers, to the home cage for 30 min for a 2 day familiarization period. Mice were then food deprived for 6, 12, or 24 h on three separate occasions spaced 5–6 days apart. The first occasion occurred a day following the 2 day familiarization period. After each food deprivation period, mice were given Ensure (1.1 kcal/g) and their intake was monitored for 60 min. Intake readings were taken every 5 min for the first 15 min and then every 15 min for the remaining 45 min. The criteria for initiating food deprivations were either no greater than a 2% change of the animals’ free feeding body weight before the experiment or 4 days since the last test.

1.2.2.

Preload study

Mice were randomly assigned to groups. For a repeated measures design, all mice progressed through four different

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conditions in a counterbalanced order. The four conditions differed in presence (P) or absence of preload (NP) and the preload to test meal interval (30 min or 60 min). Further, mice had three test sessions in each of the four conditions (P30, NP30, P60 and NP60) to control for possible conditioning effects. A test session was defined as a 12 h fast followed by a preload then a liquid (Ensure) test meal. The preload given was 1.13 ml, which was 50% of the mean volume consumed by all mice after the 12 h deprivation period in the first deprivation study. The preload was presented in a pipette as described above through the lid of the home cage. A 15 min access time to the preload was allowed to insure that the entire preload volume was consumed. The test meal lasted 30 min. Water was not available during the preload and test meal access periods. Body weights were recorded before and after the 12 h deprivation periods.

1.3.

Experiment 2: effect of CCK or BN on test meal intake

A total of 40 mice (12 WT, 12 HET and 16 KO) aged 14–23 weeks old at the beginning of the study were used. Males and females were included in all the genotypes but the relative numbers were not identical. All animals were included in the data analysis because we neither anticipated nor found obvious sex differences in the effects of the peptides. Mice were acclimated to the vivarium in the Department of Psychology in groups, and 5 days before the beginning of the experiment they were housed individually in standard shoebox cages.

1.3.1.

Procedure

All mice were first adapted to Ensure as described in Experiment 1. They had no previous experience with food deprivation. Food and water were temporarily removed during the 30 min intake adaptation trials. Separate cohorts of mice were used for the CCK and BN studies. Bombesin and sulfated CCK-8 (Sigma, St. Louis, MO) were dissolved in 0.9% physiological saline vehicle. Mice were food deprived for 12 h, and then given intraperitoneal (ip) injections of peptide or vehicle in a volume of 2 ml/kg. Five minutes after the injections, mice were given access to Ensure in their home cage for 30 min. The initial and final (30 min) readings were taken and the volume consumed calculated to the nearest 0.1 ml. Three doses each of CCK-8 (2, 6, and 18 mg/kg) and BN (2, 4 and 8 mg/kg) were used. These dose ranges were based on previous work in mice [11,16,27,35]. Doses received by the mice were administered by a modified Latin square design and saline injections were given on days between drug injections for a total of three injections each of peptide and saline. CCK and BN have short half-lives and we anticipated no carryover effects between testing days. Both peptides were thawed immediately before the injections and kept on ice for the duration of the injections. Injection of vehicle or peptide within a group was counterbalanced to control for order effects. Testing was done every 3 days to allow mice to recover initial body weights between each 12 h fast.

1.4.

Data analyses

1.4.1.

Experiment 1

In the food deprivation study, univariate ANOVAs were done comparing the effects of time point, genotype and deprivation

period on Ensure intake. In the preload study, mice that did not drink the entire preload in any of the three consecutive sessions within a condition were not included in analyses. A Student’s ttest was used to compare the last session mean within each condition to the first session mean in the next condition (i.e. P30session 3 to P60-session 1) to look for conditioning effects. Oneway ANOVAs were done to determine if there were differences between the three sessions within each treatment condition (P30, NP30, P60 and NP60). No differences were found so the three sessions were averaged and used as a grouping variable. Univariate ANOVAs were done to determine the effects of gender, genotype, preload and preload–test meal interval on Ensure intake. Body weights were compared between genotype and gender using a univariate ANOVA. For ANOVA main effects, and for Bonferroni post hoc pairwise comparisons, the significance level was set at p < .05. All statistics were computed using SPSS v. 13.0 (SPSS Inc., Chicago, IL).

1.4.2.

Experiment 2

One-way ANOVAs were used to examine main effects of gender, dose of each peptide, and genotype. Univariate ANOVAs were used to examine for interactions between the independent variables. Intake after the three ip saline treatment days were averaged and used as the comparison mean for the respective intakes post peptide administration. Student’s t-test were used to compare individual intake means after peptide administration versus saline administration. Body weights were analyzed using a one-way ANOVA. For ANOVA main effects, and for Bonferroni post hoc pairwise comparisons, the significance level was set at p < .05.

2.

Results

2.1. Experiment 1: effect of food deprivation then preload on test meal intake All mice drank Ensure during the adaptation trials. Some of the mice showed slight neophobia on the first day but by the second day all were drinking the diet without difference between genotype. Mean (S.E.) intakes were comparable in WT (1.6  0.1 ml), HET (1.6  0.1 ml) and KO (1.4  0.1 ml) mice. The highest intakes occurred in the first 5 and 10 min of the trial and most mice had stopped drinking within 15 min. Thus, 15 min was chosen for preload access time in the subsequent study. In the preliminary study to determine whether length of deprivation affected intake, we found a small but significant effect of deprivation period with the highest intake after the 24 h fast ( p < .05). There were small but not significant differences ( p = .08) between the genotypes (Table 1) and the mean intakes of all groups after all levels of deprivation

Table 1 – Mean (WS.E.) intake of Ensure (ml) after three food deprivation periods

WT HET KO

6h

12 h

24 h

2.3  0.1 2.2  0.1 2.0  0.1

2.4  0.1 2.3  0.1 2.2  0.1

2.4  0.1 2.2  0.1 2.3  0.1

peptides 27 (2006) 3226–3233

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Fig. 2 – Mean weights of mice from the preload study in experiment 1. *Genotype ( p < .001) and #gender ( p < .001) had main effects.

2.2.

Fig. 1 – Ensure intake as a function of preload condition and genotype. *No preload (NP) groups drank more than preload (P) groups ( p < .001). #Genotype had an effect ( p < .05) on intake after the 30 min preload–test meal interval (Graph A) but not after the 60 min interval (Graph B). N = 20 mice for each genotype.

varied by no more than 20%, so the intermediate deprivation time of 12 h was chosen. The results of repeated measures within each preload condition revealed no significant changes indicative of conditioning. So the data were averaged across each of the three replicate trials for each condition for analysis and presentation in Fig. 1. To streamline interpretation and analyses, data were then analyzed for main effects of preload, genotype and preload-to-test meal interval. Mice drank significantly less after a preload than no preload ( p < .001). They drank more after a 60 min than a 30 min preload-to-test interval ( p < .01) and KO mice drank slightly more than WT, though the difference did not reach significance. The average weights of mice calculated from measures taken before and after the fasts during the preload study are depicted in Fig. 2. The weights of the three genotypes differed significantly from each other ( p’s < .05) with KO weighing the most and WT the least. Males weighed more than females in all groups ( p < .001). There was also an interaction between gender and genotype that was due primarily to WT and HET groups ( p < .05).

Experiment 2: effect of CCK or BN on test meal intake

As in experiment 1, all mice were drinking Ensure reliably by the second day of adaptation without difference between genotype (data not shown). During the main phase of the experiment, CCK produced a dose-dependent reduction in test meal intake ( p < .001; Fig. 3A–C). There was also a main effect of genotype ( p < .05) with intakes of KO mice significantly lower than those of WT. Intakes of HET mice were intermediate and did not differ significantly from either WT or KO. BN administration ( p < .001) and genotype ( p < .05) had main effects on intake, but with no interaction. The anorectic effects of BN were less marked than those of CCK, but significant reductions in intake after 8 mg/kg BN relative to vehicle were observed in HET ( p < .01) and KO mice ( p < .05) (Fig. 4B and C). Intake measures after each vehicle injection were pooled and analyzed as a function of genotype. This was done separately for the mice in the CCK and in the BN study. In both cases, there were no differences in basal intake of Ensure as a function of genotype. There was an expected difference in weights with KO mice consistently heavier ( p < .05) than WT, with HET intermediate (Fig. 5).

3.

Discussion

The present experiments were designed to test the hypothesis that the hyperphagic phenotype seen in MC4RKO mice might be due to defective post ingestive feedback. Our first goal was to assess any genotype dependent response to different levels of food deprivation. MC4RKO mice did not consume more after a fast than wild type mice. This result is similar to a previous report in which food restricted MC4RKO mice ate comparable amounts to wild type mice after resumed access to food [8]. It is possible that this result is driven in part by the high palatability of the diet we used, but based on their affective responsiveness to brief-access trials of different compounds, there is no reason to suspect a genotype difference [15].

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Fig. 3 – Ensure intake during 30 min test meal after CCK administration. Horizontal lines in each graph represent the genotype vehicle mean (solid) and the standard error (dashed). Intake after CCK doses are shown as the mean W S.E. Significance from post ip vehicle intake at .05*, .01** and .001*** levels.

We then examined whether MC4RKO mice are hyperphagic due to impaired detection of gastric volume and associated calories. All genotypes tested were capable of discriminating presence or absence of a preload after a fast. MC4RKO mice drank more than the other genotypes in all conditions but this difference reached significance only in the 30 min preload–test meal interval condition. Our result differs from a recent report by Azzara et al. [5] in which KO were less sensitive to nutrientrelated signals than WT. There are several methodological differences that may account for this discrepancy. Azzara et al. [5] administered intraduodenal infusions of different nutrients while volume was held constant and in their control condition mice received equivalent acute distention of the duodenum by saline infusion. In contrast, we used the natural route of ingestion and no preload as the control condition. Thus, the differences might include cephalic phase hormonal responses and/or gastric emptying. Differences in the potency of fat or carbohydrate preloads given by oral versus post-oral routes have been shown in rats [60]. In another study [55] we have found that KO mice exhibit larger meals than WT in an operant progressive ratio closed economy environment. This is consistent with many other models of rodent hyperphagia and obesity in which a large meal phenotype is common. This suggests that once a meal is started, the signals that terminate the meal may be defective in KO mice. Two potential signals known to be involved in meal termination are CCK and BN [3,20]. Thus, we hypothesized that KO mice would be less sensitive to the satiation effects of exogenous CCK or BN than WT. Contrary to this hypothesis, we found that MC4RKO mice were at least as sensitive, and possibly more sensitive, than WT to the effects of BN and CCK. This result differs from the one report in the literature [16] that MC4RKO mice were insensitive to CCK. Methodological differences between Fan et al. [16] and the present study include dose, deprivation, test meal composition, and age. We do not believe that dose difference can account for the discrepancy because the dose of CCK used by Fan et al. (3 nmol/kg or 3.42 mg/kg) is within the range that we used and is comparable to studies in other strains of mice in which threshold anorectic doses are typically 2 mg/kg (e.g. [11,27,35,50,62]). Likewise we do not believe that the difference in deprivation level (16 h by Fan et al., compared with 12 h this study) is a significant factor because although the efficacy of CCK may differ slightly with deprivation level, this particular difference is likely to be inconsequential [12,40,49]. The difference in diet (pelleted chow by Fan et al. compared with Ensure liquid in this study) also is unlikely to be a factor because previous studies using comparable doses of CCK in mice found suppression of food intake using diets as diverse as rodent pellets, 20% sucrose, and nutritionally complete liquid diets [11,18,62]. Further, in a subsequent study in our lab, we have replicated our result using a solid diet. This leaves age as an identifiably different factor. Our study used mice aged between 14 and 23 weeks old while Fan et al. used mice at 9 weeks of age. Although both of these ages are normally considered adult in mice, age differences in CCK action have been reported previously: ob/ob mice were reported to be insensitive to CCK at 5–6 weeks of age, but showed anorexia at 7–8 weeks [35]. We cannot rule out the

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Fig. 5 – Weights of mice throughout testing in experiment 2. All genotypes differ significantly from each other at the different time points ( p < .01). *KO mice are significantly heavier than WT mice ( p < .05).

Fig. 4 – Ensure intake during 30 min test meal after BN administration. Horizontal lines in each graph represent the genotype vehicle mean (solid) and the standard error (dashed). Intake after BN doses are shown as the mean W S.E. Significance from post ip vehicle intake at .05* and .01** levels.

possibility that insensitivity to CCK is a developmental phenotype in MC4RKO mice that disappears by about 3 months of age; in this regard, age-related changes in leptin responsiveness have been reported in these mice [31]. However, the hyperphagia in MC4RKO persists well beyond 3 months of age [22]. Further studies will be needed to establish whether this hypothesized developmental change can be observed within one laboratory. One of our stated reasons for examining CCK or BN action in MC4RKO mice was to examine whether insensitivity to one or both of these peptides correlates with their hyperphagic phenotype. MC4RKO mice exhibit high endogenous leptin levels [22] and several reports have documented a synergistic relationship between leptin and CCK in reducing food intake [6,14,32,36,58,59]. Fasted rats with lower plasma levels of leptin show an attenuated anorectic effect of CCK [36] while increased leptin due to obesity can produce increased efficacy of CCK [9,34,57]. MC4RKO and HET mice showed more anorexia to BN (8 mg/ kg) than WT. Taylor and Garcia [53] found genotype dependent sensitivity in response to BN. Their ob/ob mice reduced intake at a dose of 3 nmol/kg dose but 27 nmol/kg (44 mg/kg) was required for an effect in lean mice. Our lean WT mice showed no reductions in intake from vehicle levels at any dose of BN tested, but might have shown an effect at higher doses. In this regard, BN has been found to be less potent on a molar basis than CCK [18,20]. BN receptors are found in a variety of tissues and the effect of BN on food intake may be via an interaction of both BB1 and BB2 receptors [25,37,38]. Further studies will be needed to characterize the physiological basis of the different responsiveness of WT compared with HET and KO mice. In conclusion, our experiments show that MC4RKO mice are equally or more responsive than WT mice to the effects of oral preloads of food and exogenous administration of CCK and BN on control of meal size. Some of the data reviewed suggest that obesity per se, rather than the lack of the MC4R, may be responsible for the increased responses of the KO mice to these peptides.

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Acknowledgments The stock from which these MC4RKO mice were bred was generously provided by Millennium Pharmaceuticals and this work was supported in part by NIHDK57080 (CHL).

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