CCK-8 and CCK-58 differ in their effects on nocturnal solid meal pattern in undisturbed rats

Abstract

Various molecular forms of CCK reduce food intake in rats. Although CCK-8 is the most studied form, we reported that CCK-58 is the only detectable endocrine peptide form in rats. We investigated the dark-phase rat chow intake pattern following injection of CCK-8 and CCK-58. Ad libitum-fed male Sprague-Dawley rats were intraperitoneally injected with CCK-8, CCK-58 (0.6, 1.8, and 5.2 nmol/kg), or vehicle. Food intake pattern was assessed during the dark phase using an automated weighing system that allowed continuous undisturbed monitoring of physiological eating behavior. Both CCK-8 and CCK-58 dose dependently reduced 1-h, dark-phase food intake, with an equimolar dose of 1.8 nmol being similarly effective (-49% and -44%). CCK-58 increased the latency to the first meal, whereas CCK-8 did not. The intermeal interval was reduced after CCK-8 (1.8 nmol/kg, -41%) but not after CCK-58. At this dose, CCK-8 increased the satiety ratio by 80% and CCK-58 by 160%, respectively, compared with vehicle. When behavior was assessed manually, CCK-8 reduced locomotor activity (-31%), whereas grooming behavior was increased (+59%). CCK-58 affected neither grooming nor locomotor activity. In conclusion, reduction of food intake by CCK-8 and CCK-58 is achieved by differential modulation of food intake microstructure and behavior. These data highlight the importance of studying the molecular forms of peptides that exist in vivo in tissue and circulation of the animal being studied.

Fig. 1.

CCK-58 at a dose of 1.8 nmol/kg increases the latency to eat, whereas CCK-8 does not. Rats housed under ad libitum feeding conditions with continuously open gates were injected intraperitoneally with CCK-8 (0.6, 1.8, and 5.2 nmol/kg; A), CCK-58 (0.6, 1.8, and 5.2 nmol/kg; B) or vehicle (saline containing 0.1% BSA), placed back in the cage, recording started and lights turned off. The latency to the first meal was extracted and shown for CCK-8 (A) and CCK-58 (B). Bars represent means ± SE of number of rats indicated at the bottom. *P < 0.05 vs. vehicle. #P < 0.05 vs. 0.6 nmol/kg.

Fig. 2.

CCK-8 and CCK-58 injected intraperitoneally dose-dependently decrease dark-phase food intake in rats. Gates were closed at 90 min before the onset of the dark phase. Directly before lights off, rats were intraperitoneally injected with CCK-8 (0.6, 1.8, and 5.2 nmol/kg; A), CCK-58 (0.6, 1.8, and 5.2 nmol/kg; B) or vehicle (saline containing 0.1% BSA), placed back in their home cage, the gates opened, recording started, and lights turned off. Food intake was extracted for the 1st h postinjection. Bars represent means ± SE of the number of rats indicated at the bottom. **P < 0.01 and ***P < 0.001 vs. vehicle and #P < 0.05 vs. 0.6 nmol/kg.

Fig. 3.

CCK-8 reduces the duration of the first meal, whereas CCK-58 does not. Gates were closed at 90 min before the onset of the dark phase. Directly before lights off, rats were intraperitoneally injected with CCK-8 (0.6, 1.8, and 5.2 nmol/kg), CCK-58 (0.6, 1.8, and 5.2 nmol/kg) or vehicle (saline containing 0.1% BSA), placed back in their home cage, the gates opened, recording started and lights turned off. The duration of the first meal was analyzed and shown for CCK-8 (A) and CCK-58 (B). Scale bars represent mean ± SE of number of rats indicated at the bottom. *P < 0.05 vs. vehicle.

Fig. 4.

CCK-8 and CCK-58 both reduce the meal size of the first meal and increase satiety ratio, but only CCK-8 reduces the intermeal interval. Gates were closed at 90 min before the onset of the dark phase. Directly before lights off, animals were injected intraperitoneally with CCK-8 or CCK-58 (0.6, 1.8, and 5.2 nmol/kg), or vehicle (saline containing 0.1% BSA), placed back in their home cage, the gates opened, recording started, and lights turned off. The meal size of the first meal (A, B), the intermeal interval between first and second meal of the dark phase (C, D) was extracted and the satiety ratio (intermeal interval/meal size) calculated (E, F). Bars represent mean ± SE of number of rats indicated at the bottom. *P < 0.05 and ***P < 0.001 vs. vehicle.

Fig. 5.

CCK-58, but not CCK-8, decreases the rate of ingestion of the first meal. Gates were closed at 90 min before the onset of the dark phase. Directly before lights off, rats were injected with CCK-8 (0.6, 1.8 and 5.2 nmol/kg ip), CCK-58 (0.6, 1.8 and 5.2 nmol/kg ip) or vehicle (saline containing 0.1% BSA) and placed back in their home cage. Then, the gates were opened, recording started, and lights were turned off. The rate of ingestion for the first meal was analyzed and shown for CCK-8 (A) and CCK-58 (B). Bars represent means ± SE of number of rats indicated at the bottom. **P < 0.01 vs. vehicle.

Fig. 6.

CCK-8 and CCK-58 do not reduce the meal size of the second meal. Gates were closed at 90 min before the onset of the dark phase. Directly before lights off, animals were injected with CCK-8 or CCK-58 (0.6, 1.8, and 5.2 nmol/kg ip) or vehicle (saline containing 0.1% BSA) and placed back in their home cage. Then, the gates were opened, recording started, and lights were turned off. The meal size of the second meal was extracted. Bars represent means ± SE of number of rats indicated at the bottom.

Fig. 7.

CCK-8 and CCK-58 do not induce a compensatory increase of food intake within 24 h after peptide injection. Data were analyzed over the period of 24 h following injection. There was no change in cumulative dark-phase food intake (0–12 h, A, B) or consecutive light-phase food intake (12–24 h, C, D) following injection of either CCK-8 or CCK-58, giving no indication of a compensatory increase in food intake. The cumulative 24-h food intake was not changed following injection of CCK-58 (F), whereas the highest dose of CCK-8 (E) modestly but significantly reduced the 24-h food intake. Bars represent means ± SE of number of rats indicated at the bottom. *P < 0.05 vs. vehicle and ##P < 0.01 vs. 0.6 nmol/kg.

Fig. 8.

CCK-8 increases grooming behavior and decreases locomotor activity. Rats were injected ip with CCK-8 (1.8 nmol/kg) or vehicle (saline containing 0.1% BSA) and placed back in their home cage with paper under the cage divided into six equal squares with free access to food and water. Food intake was assessed for 40 min (A). At 10 min after injection, behaviors, including eating (including food approach, B), drinking (including water approach, C), and grooming behavior (washing, licking, and scratching; D), as well as locomotor activity (total number of squares crossed; E) were monitored manually for 30 min by an observer blinded to the animals' treatment. Each behavior was counted again when lasting >5 s. Bars indicate means ± SE of 6–8 rats/group. *P < 0.05 vs. vehicle.

Fig. 9.

CCK-58 selectively decreases eating behavior, while not altering grooming and locomotor activity. Animals were injected with CCK-58 (1.8 nmol/kg ip) or vehicle (saline containing 0.1% BSA) and placed back in their home cage with paper under the cage divided into six equal squares with free access to food and water. Food intake was assessed for 40 min (A). At 10 min after injection behaviors including eating (including food approach; B), drinking (including water approach; C) and grooming behavior (washing, licking, and scratching; D), as well as locomotor activity (total number of squares crossed; E) were monitored manually for 30 min by an observer blinded to the animals' treatment. Each behavior was counted again when lasting >5 s. Bars indicate mean ± SE of 8 rats/group. *P < 0.05 vs. vehicle.