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. 2022 Apr 15;47:bjac004. doi: 10.1093/chemse/bjac004

Administration of Exendin-4 but not CCK alters lick responses and trial initiation to sucrose and intralipid during brief-access tests

Yada Treesukosol 1,✉, Timothy H Moran 2
PMCID: PMC9012268  PMID: 35427413

Abstract

Administration of cholecystokinin (CCK) or the glucagon-like peptide 1 (GLP-1) receptor agonist Exendin-4 (Ex-4) reduces food intake. Findings in the literature suggest CCK reduces intake primarily as a satiety signal whereas GLP-1 may play a role in both satiety and reward-related feeding signals. Compounds that humans describe as “sweet” and “fatty” are palatable yet are signaled via separate transduction pathways. Here, unconditioned lick responses to sucrose and intralipid were measured in a brief-access lick procedure in food-restricted male rats in response to i.p. administration of Ex-4 (3 h before test), CCK (30 min before test), or a combination of both. The current experimental design measures lick responses to water and varying concentrations of both sucrose (0.03, 0.1, and 0.5 M) and intralipid (0.2%, 2%, and 20%) during 10-s trials across a 30-min single test session. This design minimized postingestive influences. Compared with saline-injected controls, CCK (1.0, 3.0, or 6.0 µg/kg) did not change lick responses to sucrose or intralipid. Number of trials initiated and lick responses to both sucrose and intralipid were reduced in rats injected with 3.0 µg/kg, but not 1.0 µg/kg Ex-4. The supplement of CCK did not alter lick responses or trials initiated compared with Ex-4 administration alone. These findings support a role for GLP-1 but not CCK in the oral responsiveness to palatable stimuli. Furthermore, Ex-4-induced reductions were observed for both sucrose and intralipid, compounds representing “sweet” and “fat,” respectively.

Keywords: fat, sweet, appetitive, consummatory, gut peptides

Introduction

Overconsumption can be elicited by presentation of a calorically dense diet in rodents thus providing an experimental model for the overconsumption of high fat/high sugar foods that leads to diet-induced obesity in humans. The ingestive behavior in response to a calorie-dense diet can be characterized by an initial rapid rate of intake which has been referred to as the dynamic phase, followed by a static phase characterized by a plateau in food intake and weight gain change (see Brobeck 1946). Using meal pattern analysis, we previously reported that the hyperphagia observed in response to high-energy diet is driven by an increase in meal size in rats (Treesukosol and Moran 2014). The increased meal size driven hyperphagia is most robust over the first several days of high-energy diet exposure (dynamic phase). With continual diet exposure, intake and meal size decrease but remain significantly higher than those of chow-fed controls (static phase). Meal number decreases during both the dynamic and static phases. The larger but less frequent meals raise the possibilities that orosensory stimulation (e.g. taste, trigeminal, and olfactory cues) may be enhanced and/or inhibitory feedback that signals satiety and meal termination are weakened upon high calorie diet presentation.

The hyperphagia observed when animals are presented a calorie-dense diet appears to be, at least partially, driven by more immediate orosensory cues. Following sham-feeding with a sucrose solution or corn oil emulsion (nutrients consumed but immediately drained out of the stomach), rats increase food intake during subsequent 2-h tests (Tordoff and Reed 1991) supporting a role for oral cues to stimulate feeding. Compared with eating rate of standard chow, rats given a high-energy diet show high eating rate regardless of the day of presentation (Treesukosol and Moran 2014). Thus, it appears that the more immediate signals such as those coming from orosensory stimulation may have a larger influence on eating rate than long-term influences such as previous diet exposure. Electrophysiological responses of the chorda tympani nerve, a branch of the facial nerve that innervates taste receptor cells in the anterior tongue, were significantly higher to various concentrations of sweet-tasting compounds in rats maintained on a high-energy diet for 3 days, compared with responses in standard chow-fed rats (Treesukosol et al. 2018). These findings suggest short-term exposure to high-energy diet results in alterations in peripheral gustatory changes before changes in body weight and other more long-term metabolic alterations are apparent. Growing evidence points to the role of signals from the oral cavity driving ingestive behavior yet how these cues interact with postoral signals from the gut remains unclear.

A number of peptides secreted in the gastrointestinal tract influence food intake. Notable among these for the current experiment are cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1). The secretion of these peptides is altered following feeding in ways that are consistent with their roles in satiety (see Moran 2006). CCK is secreted from the duodenum in response to digestive products in the lumen and plays a role in satiation. CCK dose dependently reduces food intake through decreases in meal size (Gibbs et al. 1973; Liddle 1995) and CCK receptor antagonists drive intake by increases in meal size (Moran et al. 1993). GLP-1 is secreted from intestinal L cells in response to meal intake (Holst 1994; Chelikani et al. 2005). GLP-1 producing neurons innervate regions involved in energy homeostatic control such as hypothalamus and nucleus of the solitary tract (Han et al. 1986; Larsen et al. 1997) and expression of GLP-1 receptors has been reported in these areas (Merchenthaler et al. 1999). It has been shown that GLP-1 receptors expressed in the ventral tegmental area and nucleus accumbens, key regions of the mesolimbic pathway, are involved in hedonic drive to food intake (Dickson et al. 2012). Further evidence indicates that activation of GLP-1 receptors in the nucleus solitary tract affects food reward behavior by activity in the mesolimbic system (Richard et al. 2015). Thus, it appears that GLP-1 reduces intake via both satiety and reward-related feeding signals.

In the taste buds of the oral cavity, Type II cells express the elements for the transduction cascade for taste qualities that humans describe as “sweet,” “bitter,” and “umami.” The cells also express GLP-1 (Shin et al. 2008) and CCK (Shen et al. 2005; Herness and Zhao 2009; Yoshida et al. 2017). Application of CCK inhibits outward potassium current and induces intracellular calcium release in isolated rat taste receptor cells and this is blocked by CCK-1 receptor antagonists suggesting that taste receptor cells also express CCK receptors (Herness et al. 2002). It has been shown that CCK-like peptide in the planthopper N. lugens and the fly Drosophila mediates satiety and decreases sensitivity of gustatory neurons that express a receptor that responds to sugar (Guo et al. 2021). Furthermore, taste cell stimulation with glucose in mice has been shown to elicit increased blood GLP-1 levels (Kokrashvili et al. 2014). GLP-1 receptors are expressed on adjacent intragemmal afferent nerve fibers adjacent to taste cells (Shin et al. 2008) and GLP-1 receptor null mice have reduced peripheral electrophysiological responses to sucrose (Takai et al. 2015). In vivo, administration of a GLP-1 receptor agonist reduces intake of sucrose in rats (Mathes et al. 2012). Collectively, it appears taste receptor cells express GLP-1 and CCK and their receptors and that these are involved in taste-guided function.

Rats conditioned to avoid a high-energy diet (45% calories from fat, 17% calories from sucrose) generalized the conditioned avoidance to 100% linoleic acid and 20% intralipid and to a lesser extent 17% sucrose suggesting that the “fat” component is the more salient orosensory feature of the HE diet (Treesukosol and Moran 2018). Many compounds that are “fatty” and “sweet” are palatable to humans and are preferred by rodents, yet “fatty” and “sweet” tasting compounds are signaled via separate transduction pathways (e.g. Sclafani 2007; Mattes 2009; Cartoni et al. 2010; Liang et al. 2012; Andersen et al. 2020). It has been shown that long-chain fatty acids elicit different behavioral and physiological responses to that of medium- and short-chain fatty acids (Mattes 2009; Ruge et al. 2009). The sensations elicited by long-chain fatty acids are described by humans as unpleasant (Running et al. 2015) and thus are not consistent with “fatty” sensation of foods and fluids high in fat content. Here, palatable “fatty” sensations refer to those elicited by dietary triglycerides that when consumed target mesolimbic structures (Cansell et al. 2014) involved in reward-cue guided behaviors. Certain strains of rodents show selective preference for and more intake of “sweet” versus “fatty” compounds (Smith et al. 1994; Sclafani 2007; Sclafani et al. 2018). One of the experimental constraints of comparing responses to these compounds is how to measure responses to comparable concentrations of various stimuli. One approach has been to present a single concentration of a “sweet”-tasting compound in 1 session and a single concentration of an isocaloric “fatty” stimulus in another. The associated postingestive cues and potential testing order effects make this experimental design interpretively challenging. The experimental design employed here allows for presentation of multiple concentrations of both sweet- and fatty-tasting stimuli in a single session to investigate the effect of CCK and GLP-1 signaling on orosensory responses to these compounds.

Materials and methods

Subjects

Fourteen male Sprague-Dawley rats (Harlan) with a mean body weight of 285 g upon arrival, were single-housed in hanging wire cages in a room where humidity, temperature, and a 12–12 h light–dark cycle were automatically controlled. The rats were presented with ad libitum access to chow (2018 Teklad, Harlan) and water, except where noted. After a 7-day acclimation period to the lab environment, behavioral testing began. All procedures were approved by the Animal Care Use Committee at the Johns Hopkins University.

Procedure

Training and testing in the brief-access lick test were conducted during the light cycle in a lickometer (Davis MS-160, DiLog Instruments, Tallahassee, FL) as described previously elsewhere (e.g. Smith 2001; Glendinning et al. 2002). The rat was placed in the testing chamber of the lickometer with access to a single spout positioned ~5 mm behind a slot in the testing chamber wall. The spout was connected to a glass container holding a test stimulus. A small fan was placed above the testing chamber to direct a current of air past the spout so as to minimize potential olfactory cues from the stimuli.

During training in the lickometer, the animals were placed on a water-restricted schedule. Water access was removed from the home cages no more than 23 h before the test session. During the training period in the lickometer, fluid was only available during the daily 30-min sessions. During the first 2 training sessions, rats were presented with a stationary spout of water. Total number of licks and interlick interval were measured across the 30 min. On day 3, a shutter opened and 7 tubes of water were presented one at a time in 10-s trials. A trial was initiated when the animal licked the spout and at the end of each trial, the shutter closed. During each 8-s intertrial interval, a motorized block moved the next spout to be presented into place, after which the shutter opened and the next trial could be initiated. The rats were able to initiate as many trials as possible during each 30-min session. At the end of day 3, ad libitum access to water in the home cages resumed.

After at least 2 days with ad libitum access to chow and water, behavioral testing began. The test sessions were conducted every other day in a food-restricted condition. No more than 23 h before testing, chow was removed from the home cages. After each 30-min testing session, ad libitum chow access resumed. The test procedure involved the same session structure as day 3 but instead of 7 spouts of water, 1 spout of water, 3 concentrations of sucrose (0.03, 0.1, and 0.5 M; Sigma-Aldrich, St. Louis, MO), and 3 mixtures of intralipid (0.2%, 2%, and 20%; Sigma-Aldrich, St. Louis, MO) were presented in randomized blocks without replacement. After 3 sessions with the test compounds, animals were assigned to the saline control group or the test group such that there were no significant group differences in body weight, number of trials initiated, interlick interval values or licks to any of the test compounds. Rats in the test group were injected i.p. with Exendin-4 (Ex-4; 1.0 and 3.0 µg/kg) 3 h before the test session, CCK (1.0, 3.0, and 6.0 µg/kg) 30 min before the test session or a combination of both (1.0 µg/kg Ex-4 + 1.0 µg/kg CCK, or 3.0 µg/kg Ex-4 + 3.0 µg/kg CCK). Doses and injection times of Ex-4 and CCK were determined based on previously reported effects on chow intake (Moran et al. 1998; Emond et al. 1999; Liang et al. 2013; Yang et al. 2014). To measure if there were any carry-over effects, every other session between sessions following Ex-4/CCK injections, animals in the test group were injected with saline 30-min and/or 3 h before testing with sucrose and intralipid test array.

Data analysis

For a given animal during each test session, the mean number of licks at each stimulus concentration was calculated by collapsing all trials across the session. The mean number of licks at each concentration was compared between the control and test groups using analyses of variance (ANOVAs). The total number of trials initiated by the 2 groups was compared across the sessions. The statistical rejection criterion of 0.05 was used for all analyses.

Chow intake

To confirm that the doses of Ex-4 and CCK used were effective under these experimental conditions, their effects on chow intake were measured in the same animals following the conclusion of the brief-access lick test procedures. In line with the experimental conditions described above, animals were food restricted every other day. Rats in the test group were injected i.p. with Ex-4 (1.0 and 3.0 µg/kg) 3 h before the chow presentation, CCK (1.0, 3.0, and 6.0 µg/kg) 30 min before chow presentation or a combination of both (1.0 µg/kg Ex-4 + 1.0 µg/kg CCK, or 3.0 µg/kg Ex-4 + 3.0 µg/kg CCK). Saline control animals were injected with saline at the same time points. Chow intake following ~23-h food restriction was measured at 15, 30, 45, and 60 min after chow presentation. To measure if there were any carry-over effects, all animals were injected with saline 30-min before testing every other chow intake session (Table 1).

Table 1.

Schedule for testing sessions and injections (i.p.).

Session Test group Control group
1 Saline Saline
30 min before test session 30 min before test session
2 Saline Saline
30 min before test session 30 min before test session
3 Saline Saline
30 min before test session 30 min before test session
4 1.0 µg/kg Ex-4 Saline
3 h before test session 3 h before test session
5 Saline Saline
3 h before test session 3 h before test session
6 3.0 µg/kg Ex-4 Saline
3 h before test session 3 h before test session
7 Saline Saline
30 min before test session 30 min before test session
8 1.0 µg/kg CCK Saline
30 min before test session 30 min before test session
9 Saline Saline
30 min before test session 30 min before test session
10 3.0 µg/kg CCK Saline
30 min before test session 30 min before test session
11 Saline Saline
3 h and 30 min before test session 3 h and 30 min before test session
12 1.0 µg/kg Ex-4 3 h before test session + 1.0 µg/kg CCK Saline
30 min before test session 3 h and 30 min before test session
13 Saline Saline
3 h and 30 min before test session 3 h and 30 min before test session
14 3.0 µg/kg Ex-4 3 h before test session + 3.0 µg/kg CCK Saline
30 min before test session 3 h and 30 min before test session
15 Saline Saline
30 min before test session 30 min before test session
16 6.0 µg/kg CCK Saline
30 min before test session 30 min before test session
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Results

Exendin-4

Animals injected with 1.0 µg/kg Ex-4 and saline-injected control animals licked similarly to water (t(12) = 0.373, P = 0.715) and across the sucrose (P = 0.544) and intralipid concentrations (P = 0.157). The 2 groups also initiated similar number of trials during the test session (t(12) = 1.557, P = 0.145) (Fig. 1; Table 2).

Fig. 1.

Fig. 1.

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Mean number ± SE licks in 10-s trials to water and various concentrations of sucrose and intralipid (left panels). Mean number of trials ± SE initiated across 30-min sessions by animals injected with 1.0 μg/kg Ex-4 (black symbols, top panel) or 3.0 μg/kg Ex-4 (black symbols, bottom panel) 3 h before the session, or saline (white symbols). * indicates significant group difference P ≤ 0.05.

Table 2.

Two-way ANOVA values comparing licks to sucrose and intralipid between test and control groups.

Compared with saline-injected controls Group Concentration Group × Concentration
1.0 µg/kg Ex-4
 Sucrose F(1,12) = 0.390, P = 0.544 F(2,24) = 183.909, P < 0.001 F(2,24) = 0.129, P = 0.880
 Intralipid F(1,12) = 2.281, P = 0.157 F(2,24) = 223.023, P < 0.001 F(2,24) = 1.291, P = 0.293
3.0 µg/kg Ex-4
 Sucrose F(1,12) = 4.412, P = 0.058 F(2,24) = 290.016, P < 0.001 F(2,24) = 3.631, P = 0.042
 Intralipid F(1,12) = 21.274, P = 0.001 F(2,24) = 369.643, P < 0.001 F(2,24) = 8.280, P = 0.002
1.0 µg/kg CCK
 Sucrose F(1,12) = 0.114, P = 0.741 F(2,24) = 87.058, P < 0.001 F(2,24) = 1.891, P = 0.173
 Intralipid F(1,12) = 0.011, P = 0.918 F(2,24) = 953.427, P < 0.001 F(2,24) = 1.559, P = 0.231
3.0 µg/kg CCK
 Sucrose F(1,12) = 0.789, P = 0.392 F(2,24) = 159.244, P < 0.001 F(2,24) = 0.352, P = 0.707
 Intralipid F(1,12) = 0.006, P = 0.937 F(2,24) = 129.219, P < 0.001 F(2,24) = 2.216, P = 0.131
6.0 µg/kg CCK
 Sucrose F(1,12) = 0.445, P = 0.517 F(2,24) = 106.577, P < 0.001 F(2,24) = 0.321, P = 0.729
 Intralipid F(1,12) = 0.027, P = 0.871 F(2,24) = 101.986, P < 0.001 F(2,24) = 0.111, P = 0.896
1.0 µg/kg Ex-4 + 1.0 µg/kg CCK
 Sucrose F(1,12) = 0.073, P = 0.792 F(2,24) = 28.202, P < 0.001 F(2,24) = 0.004, P = 0.996
 Intralipid F(1,12) = 0.907, P = 0.360 F(2,24) = 166.170, P < 0.001 F(2,24) = 0.138, P = 0.872
3.0 µg/kg Ex-4 + 3.0 µg/kg CCK
 Sucrose F(1,12) = 11.066, P = 0.006 F(2,24) = 47.253, P < 0.001 F(2,24) = 0.465, P = 0.634
 Intralipid F(1,12) = 8.903, P = 0.011 F(2,24) = 98.652, P < 0.001 F(2,24) = 0.132, P = 0.877
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At the higher dose, rats injected with 3.0 µg/kg Ex-4 showed decreased responses to both sucrose and intralipid compared with saline-injected controls. A 2-way ANOVA comparing responses to sucrose between the 2 groups revealed no main effect of group (P = 0.058), a main effect of concentration (P < 0.001), and a significant interaction (P = 0.042). Rats injected with 3.0 µg/kg Ex-4 showed significantly fewer licks at 0.5 M (P = 0.006) sucrose compared with saline-injected controls. Ex-4-injected rats also licked less to 0.1 M sucrose, but this difference did not survive Bonferroni adjustment (P = 0.062). A 2-way ANOVA comparing responses to intralipid revealed a main effect of group (P = 0.001), a main effect of concentration (P < 0.001) and a significant interaction (P = 0.002). Post hoc t-tests revealed significantly fewer licks by the Ex-4-injected rats to 20% (P < 0.001) and 2% (P = 0.012) intralipid compared with controls. Rats injected with 3.0 µg/kg Ex-4 also initiated significantly fewer trials than the saline-injected controls (t(12) = 10.250, P < 0.001). The 2 groups responded similar to water (t = −1.423, P = 0.180) (Fig. 1).

Cholecystokinin

Animals injected with 1.0 µg/kg CCK and saline-injected control animals showed comparable licking behavior to water (t(12) = −1.075, P = 0.303) and across the sucrose (group (P = 0.741) and intralipid concentrations (P = 0.918). The 2 groups did not significantly differ in the number of trials initiated (t(12) = 0.381, P = 0.710) (Fig. 2).

Fig. 2.

Fig. 2.

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Mean number ± SE licks in 10-s trials to water and various concentrations of sucrose and intralipid (left panels). Mean number of trials ± SE initiated across 30-min sessions by animals injected with 1.0 μg/kg CCK (black symbols, top panel), 3.0 μg/kg CCK (black symbols, middle panel), or 6.0 μg/kg CCK (black symbols, bottom panel) 30 min before the session, or saline (white symbols).

Unlike with Ex-4, increasing the dose of CCK did not result in group differences in responses to sucrose or intralipid. Animals injected with 3.0 µg/kg CCK and saline-injected control animals showed comparable licking behavior to water (t(12) = −1.539, P = 0.150) and across the sucrose (P = 0.392) and intralipid concentrations (P = 0.937). The 2 groups did not significantly differ in the number of trials initiated (t(12) = −0.720, P = 0.486) (Fig. 2).

Similarly, animals injected with 6.0 µg/kg CCK and saline responded similar to water (t = −1.166, P = 0.266), sucrose (P = 0.517), and intralipid concentrations (P = 0.871). The 2 groups also initiated comparable number of trials (t(12) = −1.498, P = 0.160) (Fig. 2).

Combination of Ex-4 and CCK

Animals injected with the combination of 1.0 µg/kg Ex-4 and 1.0 µg/kg CCK responded similar to saline-injected controls. The 2 groups did not significantly differ in their responses to water (t(12) = 0.593, P = 0.593) or across the sucrose (P = 0.792) and intralipid concentrations (P = 0.360). Nor did the 2 groups significantly differ in the number of trials initiated across the session (t(12) = 1.593, P = 0.137) (Fig. 3).

Fig. 3.

Fig. 3.

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Mean number ± SE licks in 10-s trials to water and various concentrations of sucrose and intralipid (left panels). Mean number of trials ± SE initiated across 30-min sessions by animals injected with 1.0 μg/kg Ex-4 3 h before the session and 1.0 μg/kg CCK 30 min before the session (black symbols, top panel), or saline (white symbols). Mean number of trials ± SE initiated across 30-min sessions by animals injected with 3.0 μg/kg Ex-4 3 h before the session and 3.0 μg/kg CCK 30 min before the session (black symbols, bottom panel), or saline (white symbols). * indicates significant group difference P ≤ 0.05.

Animals injected with the combination of 3.0 µg/kg Ex-4 and 3.0 µg/kg CCK decreased responses to sucrose and intralipid. A 2-way ANOVA comparing responses to sucrose between the 2 groups revealed a main effect of group (P = 0.006), a main effect of concentration (P < 0.001) and no significant interaction (P = 0.634). Post hoc t-tests revealed significant group differences at 0.1 M (P = 0.001) and 0.5 M (P = 0.009) sucrose. A 2-way ANOVA comparing responses to intralipid between the 2 groups revealed a main effect of group (P = 0.011), a main effect of concentration (P < 0.001) and no significant interaction (P = 0.877). Post hoc t-tests revealed significant group differences at 2% (P = 0.033) and 20% (P = 0.002) intralipid. The combination of 3.0 µg/kg Ex-4 and 3.0 µg/kg CCK significantly reduced the number of trials initiated (t(12) = 10.250, P < 0.001) compared with saline (Fig. 3). The decreased response to sucrose and intralipid produced by the combination of 3.0 µg/kg Ex-4 and 3.0 µg/kg CCK was not significantly different to the decreased response elicited by 3.0 µg/kg Ex-4 alone. A 2-way ANOVA comparing responses to sucrose between the 2 conditions revealed no main effect of condition (F(1,12) = 1.365, P = 0.265), a main effect of sucrose concentration (F(2,24) = 124.765, P < 0.001), and no interaction effect (F(2,24) = 82.264, P = 0.268). Similarly, a 2-way ANOVA comparing responses to intralipid between the 2 conditions revealed no main effect of condition (F(1,12) = 0.038, P = 0.848), a main effect of intralipid concentration (F(2,24) = 215.205, P < 0.001), and no interaction effect (F(2,24) = 40.269, P = 0.458). The number of trials initiated did not significantly differ between the combination of 3.0 µg/kg Ex-4 and 3.0 µg/kg CCK or 3.0 µg/kg Ex-4 alone (t(12) = −1.243, P = 0.238).

Chow intake

Chow intake in animals following injections of Ex-4, CCK, or a combination was significantly lower than that of saline-injected controls (Fig. 4). A 2-way ANOVA comparing intake between rats injected with saline and 1.0 µg/kg Ex-4 revealed a main effect of group (P < 0.001), main effect of time (P < 0.001), and significant interaction (P < 0.001). Similarly, comparing chow intake between rats injected with saline and 3.0 µg/kg Ex-4 revealed a main effect of group (P < 0.001), main effect of time (P < 0.001), and a significant interaction (P < 0.001) (Table 3).

Fig. 4.

Fig. 4.

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Mean cumulative chow intake ± SE following ~23 h food restriction and injections of Ex-4 3 h before chow presentation, CCK, 30 min before chow presentation or saline control. * indicates significant group difference at given time points P ≤ 0.05.

Table 3.

Two-way ANOVA values comparing chow intake between test and control groups.

Compared with saline-injected controls Group Time Group × Concentration
1.0 µg/kg Ex-4 F(1,12) = 24.044, P < 0.001 F(3,36) = 96.088, P < 0.001 F(3,36) = 9.853, P < 0.001
3.0 µg/kg Ex-4 F(1,12) = 70.387, P < 0.001 F(3,36) = 54.955, P < 0.001 F(3,36) = 10.298, P < 0.001
1.0 µg/kg CCK F(1,12) = 3.961, P = 0.070 F(3,36) = 51.267, P < 0.001 F(3,36) = 1.384, P = 0.263
3.0 µg/kg CCK F(1,12) = 3.046, P = 0.106 F(3,36) = 57.937, P < 0.001 F(3,36) = 1.298, P = 0.290
6.0 µg/kg CCK F(1,12) = 5.054, P = 0.044 F(3,36) = 38.252, P < 0.001 F(3,36) = 0.110, P = 0.953
1.0 µg/kg Ex-4 + 1.0 µg/kg CCK F(1,12) = 29.506, P < 0.001 F(3,36) = 77.704, P < 0.001 F(3,36) = 6.110, P = 0.002
3.0 µg/kg Ex-4 + 3.0 µg/kg CCK F(1,12) = 78.351, P < 0.001 F(3,36) = 114.886, P < 0.001 F(3,36) = 28.327, P < 0.001
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Tested in food-restricted conditions, CCK had a weaker effect of chow intake suppression than Ex-4. A 2-way ANOVA comparing chow intake between saline- and 1.0 µg/kg CCK-injected rats did not reveal a main effect of group (P = 0.070), revealed a main effect of time (P < 0.001), and no significant interaction (P = 0.263). Similarly, a 2-way ANOVA comparing chow intake between saline- and 3.0 µg/kg CCK-injected rats revealed no main effect of group (P = 0.106), a main effect of time (P < 0.001), and no interaction effect (P = 0.290). However, chow intake was significantly lower compared with saline at the higher 6.0 µg/kg CCK dose. The analysis revealed a main effect of group (P = 0.044), main effect of time (P < 0.001), and no interaction effect (P = 0.953).

The combination of Ex-4 and CCK significantly reduced intake compared with saline controls. A 2-way ANOVA comparing intake between 1.0 µg/kg Ex-4 and 1.0 µg/kg CCK and saline-injected groups revealed a main effect of group (P < 0.001), a main effect of time (P < 0.001), and interaction effect (P = 0.002). Similarly, a 2-way ANOVA comparing intake between 3.0 µg/kg Ex-4 and 3.0 µg/kg CCK and saline-injected groups revealed a main effect of injection (P < 0.001), main effect of time (P < 0.001), and an interaction effect (P < 0.001). At the lower dose, intake following the combination did not significantly differ to that following Ex-4 alone. A 2-way ANOVA comparing intake between the combination of 1.0 µg/kg Ex-4 and 1.0 µg/kg CCK with 1.0 µg/kg Ex-4 alone revealed no main effect of condition (F(1,12) = 0.014), P = 0.907), a main effect of time (F(3,36) = 56.560, P < 0.001), and no interaction effect (F(3,36) = 0.202, P = 0.895). In contrast, at the higher dose the combination reduced intake more so than Ex-4 alone. A 2-way ANOVA comparing intake between the combination of 3.0 µg/kg Ex-4 and 3.0 µg/kg CCK with 3.0 µg/kg Ex-4 alone revealed a main effect of condition (F(1,12) = 56.800), P < 0.001), a main effect of time (F(3,36) = 5.656, P = 0.003), and an interaction effect (F(3,36) = 16.135, P < 0.001).

Saline control sessions

Rats injected with 3.0 µg/kg Ex-4 initiated fewer trials and showed decreased responses to both sucrose and intralipid compared with saline-injected controls. This decrease was not observed during the saline control sessions interspersed between Ex-4 and/or CCK test sessions indicating that there were no carry-over effects.

Discussion

The current findings provide evidence for a role of GLP-1, but not CCK in the oral responsiveness to palatable stimuli. Injections of 3.0 µg/kg Ex-4 decreased licking responses to sucrose and intralipid and also decreased the number of trials initiated. In contrast, compared with controls, administration of 3.0 µg/kg CCK, or even a higher dose (6.0 µg/kg), did not significantly change lick responses or trials initiated. Despite differences in effectiveness between Ex-4 and CCK to reduce responses to sucrose and intralipid, all doses of Ex-4 and the highest dose of CCK significantly reduce intake of standard chow. These findings support a role of GLP-1 but not CCK signaling, in both the appetitive and consummatory components of behavior toward sweet and fatty stimuli.

Many compounds that humans describe as “sweet” and “fatty” are palatable and calorically dense yet are signaled via separate transduction pathways. It has been previously reported, that disruption of the GLP1r gene (−/−) in mice resulted in reduced intake of 62 mM sucrose yet no significant differences from controls in response to 200 µM α-linoleic acid (Martin et al. 2012) suggesting GLP-1 plays a role in “sweet” but not “fatty” licking and intake. Here, Ex-4-induced reductions were observed for both sucrose and intralipid that represent “sweet” and “fatty” stimuli, respectively, suggesting GLP-1 plays a role in the oral responsiveness of both categories of palatable compounds. Thus, it is possible that GLP-1 plays differential roles in mice and rats thus leading to these disparate results. Another possible explanation is length of stimulus presentation across studies. In most reports in the literature, a single concentration of a stimulus is presented in a single session. The experimental design employed in the current study allows for comparisons of responses to various concentrations of both sucrose and intralipid in the same test session thus minimizing postingestive influences and test order effects. A third potential explanation is the different energy homeostatic states of the animals. In a previous study, presenting rats with varying concentrations of sucrose in brief-access lick tests, 1.0 µg/kg Ex-4 reduced the number of trials initiated when rats were nondeprived but this was not observed when rats were tested following ~23 h of no food (Mathes et al. 2012). In the current study, rats were tested in a ~23-h food-restricted state. Compared with saline-injected controls, 1.0 µg/kg Ex-4 did not significantly decrease number of trials but 3.0 µg/kg Ex-4 did. These influencing factors are not mutually exclusive. It appears that combined with energy homeostatic controls, GLP-1 signaling influences oral responses to palatable stimuli.

The role of CCK in responses driven by oral cues is also influenced by energy homeostatic state. It has been previously demonstrated that OLETF rats lacking CCK receptors drink more oil in 60-min real feeding and sham-feeding (in which ingested contents are prevented from accumulating in the stomach and small intestine) conditions compared with controls (Swartz et al. 2010) providing evidence for the role of CCK in responses to oil. Furthermore, CCK administration has been shown to reduce intake of water and a range of sucrose concentrations in satiated rats tested in a 9-min 1-bottle test. Yet when tested in a food-restricted state, CCK reduced intake of water and only the midrange sucrose concentrations (Gosnell and Hsiao 1984). Thus, it appears that CCK and energy homeostatic state influences responses to sucrose in an intake test in which oral and postoral cues drive responses. When postoral cues were minimized in 10-s presentations of various sucrose solutions, OLETF rats licked more than controls to some mid- and high-range concentrations (Hajnal et al. 2005), yet here CCK did not significantly affect unconditioned lick responses to sucrose and intralipid. A possible explanation for these seemingly disparate findings may be partially attributed to other compensatory changes in the CCK receptor lacking OLETF rats such as in differences in responses to water compared with control rats, that are not evident following exogenous CCK administration. Another point of consideration is the experimental design employed in many published findings in which a fixed number of trials is presented to all subjects. In this way, it can be difficult to discern whether a lack of responses is driven by appetitive or consummatory components of behaviors. In the current experiment, animals initiated each trial thus providing measures that can segregate some of these behavioral components. Here, CCK did not change lick responses (consummatory), nor number of trials initiated during these test sessions.

In the current study, even at a high dose of 6.0 µg/kg which suppressed chow intake under food-restricted conditions, CCK did not affect trials initiated or lick responses to sucrose and intralipid presentations. One explanation for these findings is that the role of CCK in taste transduction may be limited to bitter-tasting compounds. In taste buds, a subset of taste cells express CCK (Herness et al. 2002) and CCK receptors (Herness and Zhao 2009; Yoshida et al. 2017). Many of these CCK-expressing cells coexpress Gα-gustducin involved in the transduction of compounds that human describe as umami, sweet, and bitter. Yet of these cells, only a small subset also express T1R3 (Herness et al. 2005; Yoshida et al. 2017) which is a component of the receptor for the transduction of “umami” and “sweet” tastes. Thus, it is unlikely that CCK-expressing cells in the taste bud are involved with sweet-guided oral responses, rather it is plausible that the majority of these cells are involved with “bitter” taste. In support of this hypothesis, many CCK-sensitive taste cells show calcium responses to compounds described by humans as bitter (quinine and caffeine) (Lu et al. 2003). Further, CCK KO mice show reduced neural responses to bitter compounds but normal responses to sucrose (and other taste compounds) (Yoshida et al. 2017). Taken together it appears that CCK does not play a role in sweet or fatty oral-guided responses.

Although the actions of various gut peptides in the control of ingestive behavior have been individually studied, ingestion does not alter the secretion of only a single gut peptide. A number of studies have examined the potential for synergistic effects among gut peptides. Combination injections of individual doses produce decreases in feeding that are more than the sum of their individual effects (Hinton et al. 1986; Le Sauter and Geary 1987). Here, the combination of CCK and Ex-4 reduced intake of chow more so than 3.0 µg/kg Ex-4 or 3.0 µg/kg CCK alone, yet compared with individual peptides, the combination did not differentially affect licking responses or trials initiated to sucrose or intralipid.

The doses of Ex-4 and CCK administered were chosen based on effects on chow intake (Moran et al. 1998; Emond et al. 1999; Liang et al. 2013; Yang et al. 2014). To motivate responses in the current series of brief-access taste tests, rats were tested in a food-restricted condition. As such, chow intake was measured under the same experimental conditions including dose and injection times of Ex-4 and CCK and testing every other day under food-restriction states. Although all doses used also reduced chow intake, the lower doses of CCK did not significantly reduce chow intake at all time points tested. This is consistent with previous reports in which food deprivation schedules blunt the food suppression effects of CCK in rats (McMinn et al. 2000) and baboons (Stein et al. 1986) which appears to at least be partially mediated by noradrenergic A2 neurons centrally (Maniscalco and Rinaman 2013). The role of these gut peptides in oral and postoral signals is influenced by energy homeostasis.

The current experimental design provides a means to compare responses to various concentrations of both sucrose and intralipid in a single test session. Here, doses of CCK and Ex-4 that decrease chow intake differentially affected responses to sucrose and intralipid. Although “sweet” and “fatty” compounds signal separate transduction pathways and have different influences in intake, Ex-4 administration reduced responses to both. Taken together, the findings support a role of GLP-1 but not CCK, in orosensory-guided behavior toward palatable stimuli.

Acknowledgements

Parts of this paper were presented at the 24th Annual Meeting of the Society for the Study of Ingestive Behavior, Port, Portugal, July 2016.

Funding

This work was supported by the National Institutes of Health [grant number DK019302 to THM].

Conflict of interest

None declared.

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