Normal feeding and body weight in Fischer 344 rats lacking the cholecystokinin-1 receptor gene

Abstract

A large body of evidence has demonstrated that one mechanism by which cholecystokinin (CCK) inhibits food intake through activation of CCK1 receptors (CCK1R) on vagal afferent neurons that innervate the gastrointestinal tract and project to the hindbrain. OLETF rats, which carry a spontaneous null mutation of the CCK1R, are hyperphagic, obese, and predisposed to type 2 diabetes. Recently, by introgressing the OLETF-derived, CCK1R-null gene onto a Fischer 344 genetic background, we have been able to generate a CCK1R-deficient, congenic rat strain, F344.Cck1r(-/-), that in contrast to OLETF rats, possesses a lean and normoglycemic phenotype. In the present study, the behavioral and neurobiological phenotype of this rat strain was characterized more fully. As expected, intraperitoneal injections of CCK-8 inhibited intake of chow and Ensure Plus and induced Fos responses in the area postrema and the gelatinosus, commissural and medial subdivisions of the nucleus tractus solitarius of wild-type F344.Cck1r(+/+) rats, whereas CCK-8 was without effect on food intake or Fos induction in the F344.Cck1r(-/-) rats. F344.Cck1r(-/-) and F344.Cck1r(+/+) rats did not differ in body weight and showed comparable weight gain when maintained on Ensure Plus for 2 weeks. Also, no difference was found in 24-h food intake, and dark-phase meal frequency or meal size between F344.Cck1r(+/+) and F344.Cck1r(-/-) rats. As expected, blockade of endogenous CCK action at CCK1R increased food intake and blocked the effects of peripheral CCK-8 in wild-type F344.Cck1r(+/+) rats. These results confirm that in rats with a F344 background, CCK-1R mediates CCK-8-induced inhibition of food intake and Fos activation in the hindbrain and demonstrate that selective genetic ablation of CCK1R is not associated with altered meal patterns, hyperphagia, or excessive weight gain on a palatable diet.

Figure 1.. Genotyping of Chromosome 14. STS/SSLP marker map of chromosome 14.

The new availability of high density markers from the Rat Genome Database expanding the size of chromosomes allow us to define more precisely the fragment introgressed onto F344 rat to 37-Mb. The UCSC map marker location and the size of the polymorphic marker between the two parental strains OLETF and F344 are shown. The numerical color code is: yellow (1) OLETF, blue (2) F344, and yellow-blue (2–1-2) F344.Cck1r congenic line carrying a DNA fragment of 37 Mb from OLETF rat.

Figure 2A-B.. Effects of CCK-8 on Chow Intake in Mutant F344.CCK1r^−/− and Wild-type F344.CCK1r^+/+ Rats :

CCK-8 (2 nmol/kg), as expected, inhibited food intake at 30 min by 87% in F344.Cck1r^+/+ (Figure 2A), whereas CCK-8 at this dose was ineffective at inhibiting food intake in F344.Cck1r^−/− rats (Figure 2A). Similarly, the higher dose of CCK-8 (8 nmol/kg) inhibited food intake at 30 min by 97% in F344.Cck1r^+/+ whereas in F344.Cck1r^−/− rats, CCK-8 did not inhibit food intake (Figure 2B).

Figure 3.. Effects of CCK-8 on 30-minute intake of Ensure Plus Vanilla in Mutant F344.Cck1r^−/− and Wild-type F344.Cck1r^+/+ Rats.

CCK-8 (8 nmol/kg) inhibited Ensure intake at 30 min by 87% in F344.Cck1r^+/+ rats, but failed to alter food intake F344.Cck1r^−/− rats.

Figure 4A-D.. Effects of CCK-8 on Fos induction in the Area Postrema in Mutant F344.Cck1r^−/− and Wild-type F344.Cck1r^+/+ Rats.

Fos activation is revealed by concentration of immunoreactive Fos in cell nuclei (Fos (+) cells) in the AP. Fos-immunostaining was done by Cy3 fluorescence. Images were taken from the whole AP. Figure 4A: Peripheral injection of vehicle induced little or no Fos (+) neurons in the AP of F344.Cck1r^+/+ rats. Figure 4B: Peripheral injection of CCK-8 (8 nmol/kg) induced numerous Fos (+) neurons in the AP of F344.Cck1r^+/+ rats. Figure 4C: Peripheral injection of vehicle induced little or no Fos (+) neurons in the AP of F344.Cck1r^−/− rats. Figure 4D: Peripheral injection of CCK-8 (8 nmol/kg) induced little or no Fos (+) neurons in the AP of F344.Cck1r^−/− rats. A-D all visualized at 20X magnification.

Figure 5A-B.. Quantitative Effects of CCK-8 on Fos induction in the AP, cmNTS, cNTS, and gNTS in Mutant F344.Cck1r^−/− and Wild-type F344.Cck1r^+/+ Rats.

CCK-8 (8 nmol/kg), as expected, stimulated Fos induction in the AP, cmNTS, cNTS, and gNTS (AP, cmNTS, cNTS, and gNTS noted on Y axis labels) by 5237, 1931, 13757, and 5471% in F344.Cck1r^+/+ (Figure 4A), but was without effect in F344.Cck1r^−/− (Figure 5B). As can be observed by the scale on the Y axis between Figures 5A-B, the absolute magnitude of CCK-8-elicited Fos induction in the hindbrain was nearly 20–50-fold greater in F344.Cck1r^+/+ vs. F344.Cck1r^−/− rats.

Figure 6A-D.. Effects of CCK-8 on Fos induction in the cmNTS in Mutant F344.Cck1r^−/− and Wild-type F344.Cck1r^+/+ Rats.

Fos activation is revealed by concentration of immunoreactive Fos in cell nuclei (Fos (+) cells) in the cmNTS. Fos-immunostaining was done by Cy3 fluorescence. Images were taken from the right side of the cmNTS with the AP shown in the upper left. Figure 6A: Peripheral injection of vehicle induced little or no Fos (+) neurons in the cmNTS of F344.Cck1r^+/+ rats. Figure 6B: Peripheral injection of CCK-8 (8 nmol/kg) induced numerous Fos (+) neurons in the cmNTS of F344.Cck1r^+/+ rats. Figure 6C: Peripheral injection of vehicle induced little or no Fos (+) neurons in the cmNTS of F344.Cck1r^−/− rats. Figure 6D: Peripheral injection of CCK-8 (8 nmol/kg) induced little or no Fos (+) neurons in the cmNTS of F344.Cck1r^−/− rats. A-C all visualized at 20X magnification.

Figure 7A-B.. Meal number and meal size in 6 h fasted and ad libitum fed Mutant F344.Cck1r^−/− and Wild-type F344.Cck1r^+/+ Rats.

Meal number was not different at 1, 4, or 12 hours after provision of food between F344.Cck1r^+/+ and F344.Cck1r^−/− rats following a 6-h fast or when fed Ensure Plus ad libitum (Figure 7A). Meal size was not different at 1, 4, or 12 hours after provision of food between F344.Cck1r^+/+ and F344.Cck1r^−/− rats following a 6-h fast or when fed Ensure Plus ad libitum (Figure 7B).

Figure 8.. Body weight gain following 2-week exposure to Ensure Plus Vanilla in Mutant F344.Cck1r^−/− and Wild-type F344.Cck1r^+/+ Rats.

Body weight in animals (n=8/genotype) was monitored before and after a 2-week exposure to Ensure Plus Vanilla. Both genotypes gained a comparable amount of weight: (F344.Cck1r^−/− (65.5 ± 2.6 g) vs. F344.Cck1r^+/+ (61.8 ± 4.5 g)). There also was no difference in body weights between groups at the start or completion of exposure to Ensure Plus Vanilla.

Figure 9.. Response of Wild-type F344.Cck1r^+/+ Rats to Endogenous CCK1R Blockade.

Figure 9A. Devazepide (1 mg/kg) or vehicle (10% DMSO, 10% Tween 80, 80% saline) was administered to 30-min. fasted animals at 30 min. prior to the start of the dark cycle and access to food. Figure 9B. Devazepide (1 mg/kg) or vehicle was administered to 6-h fasted animals 30 min. prior to CCK-8 (8 nmol/kg) or vehicle (0.1% BSA, saline). CCK-8 was administered immediately prior to the dark cycle and access to food. Food intake was measured at 0.5, 1, 2, 3, 4, and 24 h after access to food and the start of the dark cycle for studies described in Figure 9A and 9B.