Peripherally injected CCK-8S activates CART positive neurons of the paraventricular nucleus in rats.

Cholecystokinin (CCK) plays a role in the short-term inhibition of food intake. Cocaine- and amphetamine-regulated transcript (CART) peptide has been observed in neurons of the paraventricular nucleus (PVN). It has been reported that intracerebroventricular injection of CART peptide inhibits food intake in rodents. The aim of the study was to determine whether intraperitoneally (ip) injected CCK-8S affects neuronal activity of PVN-CART neurons. Ad libitum fed male Sprague-Dawley rats received 6 or 10 microg/kg CCK-8S or 0.15M NaCl ip (n=4/group). The number of c-Fos-immunoreactive neurons was determined in the PVN, arcuate nucleus (ARC), and the nucleus of the solitary tract (NTS). CCK-8S dose-dependently increased the number of c-Fos-immunoreactive neurons in the PVN (mean+/-SEM: 102+/-6 vs. 150+/-5 neurons/section, p<0.05) and compared to vehicle treated rats (18+/-7, p<0.05 vs. 6 and 10 microg/kg CCK-8S). CCK-8S at both doses induced an increase in the number of c-Fos-immunoreactive neurons in the NTS (65+/-13, p<0.05, and 182+/-16, p<0.05). No effect on the number of c-Fos neurons was observed in the ARC. Immunostaining for CART and c-Fos revealed a dose-dependent increase of activated CART neurons (19+/-3 vs. 29+/-7; p<0.05), only few activated CART neuron were observed in the vehicle group (1+/-0). The present observation shows that CCK-8S injected ip induces an increase in neuronal activity in PVN-CART neurons and suggests that CART neurons in the PVN may play a role in the mediation of peripheral CCK-8S's anorexigenic effects.

CCK-8S activates c-Fos in a dose-dependent manner in nesfatin-1 immunoreactive neurons in the paraventricular nucleus of the hypothalamus and in the nucleus of the solitary tract of the brainstem.

Recently, a new neuropeptide, named nesfatin-1, was discovered. It has been reported that nesfatin-1 inhibits food intake after injection into the third ventricle as well as intraperitoneal (ip) injection. Cholecystokinin (CCK) is well established to play a role in the regulation of food intake. The aim of the study was to examine whether CCK-8S injected ip modulates neuronal activity in nesfatin-1 immunoreactive (ir) neurons localized in the PVN and in the nucleus of the solitary tract (NTS). Additionally, tyrosine hydroxylase-immunoreactivity (TH-ir) in the PVN was determined to assess the distribution of TH-ir fibers in relation to nesfatin-1-ir. Non-fasted male Sprague-Dawley rats received 6 or 10 microg CCK-8S/kg or vehicle solution (0.15M NaCl; n=4 all groups) ip. The number of c-Fos-ir neurons was determined in the PVN, arcuate nucleus (ARC), and NTS. Double staining procedure for nesfatin-1 and c-Fos revealed that CCK-8S increased significantly and in a dose-dependent manner the number of c-Fos positive nesfatin-1-ir neurons in the PVN ( approximately 4-fold and approximately 7-fold) and NTS ( approximately 9-fold and approximately 26-fold). Triple staining in the PVN showed a dose-dependent neuronal activation of nesfatin-1 neurons that were colocalized with CRF and oxytocin. Double labeling against nesfatin-1 and TH revealed that nefatin-1-ir neurons were encircled in a network of TH-ir fibers in the PVN. No effect on the number of c-Fos-ir neurons was observed in the ARC. These results suggest that the effects of CCK on the HPA axis and on food intake may, at least in part, be mediated by nesfatin-1-ir neurons in the PVN.

Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats.

Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13 microg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and 127 microg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12h. Ghrelin and desacyl ghrelin (64 microg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.

Peripheral obestatin has no effect on feeding behavior and brain Fos expression in rodents.

Obestatin is produced in the stomach from proghrelin by post-translational cleavage. The initial report claimed anorexigenic effects of obestatin in mice. Contrasting studies indicated no effect of obestatin on food intake (FI). We investigated influences of metabolic state (fed/fasted), environmental factors (dark/light phase) and brain Fos response to intraperitoneal (ip) obestatin in rats, and used the protocol from the original study assessing obestatin effects in mice. FI was determined in male rats injected ip before onset of dark or light phase, with obestatin (1 or 5 micromol/kg), CCK8S (3.5 nmol/kg) or 0.15 M NaCl, after fasting (16 h, n=8/group) or ad libitum (n=10-14/group) food intake. Fos expression in hypothalamic and brainstem nuclei was examined in freely fed rats 90 min after obestatin (5 micromol/kg), CCK8S (1.75 nmol/kg) or 0.15 M NaCl (n=4/group). Additionally, fasted mice were injected ip with obestatin (1 micromol/kg) or urocortin 1 (2 nmol/kg) 15 min before food presentation. No effect on FI was observed after obestatin administration during the light and dark phase under both metabolic conditions while CCK8S reduced FI irrespectively of the conditions. The number of Fos positive neurons was not modified by obestatin while CCK8S increased Fos expression in selective brain nuclei. Obestatin did not influence the refeeding response to a fast in mice, while urocortin was effective. Therefore, peripheral obestatin has no effect on FI under various experimental conditions and did not induce Fos in relevant central neuronal circuitries modulating feeding in rodents.

Peripheral injection of ghrelin induces Fos expression in the dorsomedial hypothalamic nucleus in rats.

Peripheral ghrelin has been shown to act as a gut-brain peptide exerting a potent orexigenic effect on food intake. The dorsomedial nucleus of the hypothalamus (DMH) is innervated by projections from other brain areas being part of the network of nuclei controlling energy homeostasis, among others NPY/AgRP-positive fibers arising from the arcuate nucleus (ARC). The aim of the study was to determine if peripherally administered ghrelin affects neuronal activity in the DMH, as assessed by Fos expression. The number of Fos positive neurons was determined in the DMH, paraventricular nucleus of the hypothalamus (PVN), ARC, ventromedial hypothalamic nucleus (VMH), nucleus of the solitary tract (NTS) and in the area postrema (AP) in non-fasted Sprague-Dawley rats in response to intraperitoneally (ip) injected ghrelin (3 nmol/rat) or vehicle (0.15 M NaCl). Peripheral ghrelin induced a significant increase in the number of Fos-ir positive neurons/section compared with vehicle in the ARC (mean+/-SEM: 49+/-2 vs. 23+/-2 neurons/section, p=0.001), PVN (69+/-5 vs. 34+/-3, p=0.001), and DMH (142+/-5 vs. 83+/-5, p<0.001). Fos-ir positive neurons were mainly localized within the ventral part of the DMH. No change in Fos expression was observed in the VMH (53+/-8 vs. 48+/-6, p=0.581), NTS (42+/-2 vs. 40+/-3, p=0.603), and in the AP (7+/-1 vs. 5+/-1, p=0.096). Additional double-labelling with anti-Fos and anti-AgRP revealed that Fos positive neurons in the DMH were encircled by a network of AgRP-ir positive fibers. These data indicate that peripheral ghrelin activates DMH neurons and that NPY-/AgRP-positive fibers may be involved in the response.