Ayman I. Sayegh

Cholecystokinin activates specific enteric neurons in the rat small intestine.

Cholecystokinin (CCK) is a peptide hormone released from the I-cells of the upper small intestine. CCK evokes a variety of physiological responses, such as stimulation of pancreatic secretion, reduction of food intake and inhibition of gastric emptying. Previously, we reported that CCK activates enteric neurons in the rat. However the specific subpopulations of enteric neurons activated by CCK have not been identified. In the work reported here, we utilized immunohistochemical detection of nuclear Fos, a marker for neuronal activation, and selected phenotypic markers to identify some of the neuronal subpopulations activated by CCK. The phenotypic markers that we examined were: nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calbindin (Cal), Calretinin (Calr), and neurofilament-M (NF-M). We found that in the myenteric plexus of the rat duodenum and jejunum, CCK activated NOS immunoreactive neurons. In the submucosal plexus of duodenum and jejunum, CCK activated Cal, Calr and NF-M immunoreactive neurons. CCK failed to activate NK-1R immunoreactive neurons in either plexus. Our results indicate that CCK activates distinct enteric neurons in the rat upper small intestine. Furthermore the fact that NOS immunoreactive neurons were activated suggests that CCK modulates the activity of inhibitory motor neurons in the myenteric plexus. Expression of Fos immunoreactivity in Calr and Cal immunoreactive neurons is consistent with a role for CCK in modulation of intrinsic sensory and/or secretomotor neuronal activity in the submucosal plexus.

Exenatide reduces food intake and activates the enteric nervous system of the gastrointestinal tract and the dorsal vagal complex of the hindbrain in the rat by a GLP-1 receptor

UNLABELLED Exenatide is a synthetic agonist of the glucagon-like peptide-1 (GLP-1) receptor, which has also been shown to reduce food intake. The goal of this work is to test the hypothesis that exenatide reduces food intake and activates the enteric nervous system (ENS; myenteric and submucosal plexuses) of the gastrointestinal (GI) tract and the areas of the dorsal vagal complex (DVC) of the hindbrain that control food intake. EXPERIMENT 1: Five groups of overnight food-deprived male Sprague Dawley rats were injected with exenatide (0.1, 0.5, 5 and 10 microg/kg) or saline intraperitoneally, and the intake of 10% sucrose solution was measured at 5 min intervals for 120 min. All doses of exenatide reduced sucrose intake following the 20 min time point, and pretreatment with exendin (9-39), a GLP-1 receptor antagonist, reversed this reduction. EXPERIMENTS 2 AND 3: Following overnight food deprivation, five groups of rats were injected with the treatments listed above and sacrificed 90 min following the injections. The myenteric and submucosal plexuses and DVC were processed for detection of Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation). Exenatide increased Fos-LI dose-dependently in the myenteric and submucosal neurons of the duodenum, but not jejunum and ileum, and in the areas of the DVC that regulate food intake e.g. area postrema, nucleus tractus solitaries and dorsal motor nucleus of the vagus. In addition, pretreatment with exendin (9-39) prior to exenatide injection blocked the activation in both locations. CONCLUSIONS Activation of the enteric neurons by exenatide may be part of the pathway by which this peptide reduces food intake.

The Role of Cholecystokinin Receptors in the Short-Term Control of Food Intake

Cholecystokinin (CCK) is a hormone secreted by the I-cells of the upper small intestine in response to fat, protein, and some nonnutrients, for example, camostat, and a peptide/neurotransmitter secreted by neurons of the central and peripheral nervous systems. There are multiple molecular forms of CCK, for example, CCK-8, CCK-33, and CCK-58, with an active site located within the first eight amino acids of the carboxyl terminus and with a sulfate group on the seventh tyrosine residue. Physiologically, CCK increases pancreatic secretions and gallbladder and smooth muscle contractions as well as inhibits gastric emptying and food intake. CCK evokes these responses by activating two G protein-coupled receptors: CCK(1) and CCK(2). CCK(1) receptors are located mainly in the alimentary tract and contain two affinity states, high and low, whereas CCK(2) receptors are found mainly in the brain. Although a CCK-mediated reduction in cumulative food intake occurs by the activation of low-affinity CCK(1) receptors located on vagal afferents, the vagus, and the splanchnic nerves are necessary for the reduction of meal size (MS) and the prolongation of the inter-meal interval (IMI) by CCK. Finally, the reduction of food intake by CCK occurs by three possible modes of action: paracrine, endocrine, and neurocrine; thus far, the data favor a paracrine mode. In addition, the gut, which is the main source of peripheral CCK, contains the first neuronal component that senses the presence of food, the enteric nervous system. The enteric nervous system may have a role in the reduction of MS and the prolongation of the IMI by CCK.

Intestinal infusions of oleate and glucose activate distinct enteric neurons in the rat.

Nutrients entering the small intestine trigger a variety of neural and endocrine reflexes that involve specific afferents, efferents and interneurons, many of which are located within the enteric nervous system (ENS). We hypothesized that intestinal nutrient stimuli might activate specific subpopulations of these neurons. To test this hypothesis, we utilized immunohistochemical detection of nuclear c-fos expression in the myenteric and submucosal plexuses of the rat small intestine following intraintestinal infusions of oleate or glucose. Additionally, we used dual label methods to detect both Fos-immunoreactivity and immunoreactivity for five phenotypic neuronal markers: neurokinin-1 receptor (NK-1R), neurofilament-M (NF-M), neuronal nitric oxide synthase (NOS), calbindin (Cal) and calretinin (Calr), to characterize neurons that were activated by intestinal infusion of oleate and glucose. We found that oleate and glucose activated myenteric neurons in the duodenum and jejunum, but not the ileum. Oleate and glucose infusions significantly increased the number of Fos-immunoreactive nuclei in the submucosal plexus of the duodenum and jejunum, however, only glucose increased Fos-immunoreactivity in the ileum. Oleate and glucose infusions were associated with a small increase in Fos-immunoreactivity in NOS-immunoreactive neurons in the myenteric plexus. In the submucosal plexus, the majority of neurons activated by intestinal infusion of oleate or glucose were immunoreactive to Cal and Calr. In the rat, many of these neurons have Dogiel Type II-like morphology, which is consistent with the possibility that these neurons function as mucosal afferents in reflexes activated by nutrient stimuli.

Cholecystokinin-8 increases Fos-like immunoreactivity in the brainstem and myenteric neurons of rats through CCK1 receptors.

To examine the role of cholecystokinin1 receptor (CCK1) in the activation of brainstem and myenteric neurons by CCK, we compared the ability of exogenous CCK-8 to induce Fos-like immunoreactivity (Fos-LI) in these neurons in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, lacking CCK1 receptors, and Long-Evans Tokushima Otsuka (LETO) controls. Five groups (n=4 rats per group) of OLETF rats, and five LETO control groups, were injected intraperitoneally (IP) with 5, 10, 20, and 40 microg/kg CCK-8 or saline. Forty-micrometer brainstem sections containing the area postrema, nucleus of the solitary tract, and the dorsal motor nucleus of the vagus, and myenteric neurons of the duodenum, jejunum, and ileum underwent a diaminobenzidine reaction enhanced with nickel to reveal Fos-LI. CCK-8 did not increase Fos-LI in any of the tested neurons in the OLETF rats. CCK-8 increased Fos-LI in the brainstem of the LETO rats in a dose dependent manner. In the LETO rats only 40 microg/kg CCK-8 increased Fos-LI in the myenteric plexus of the jejunum. This study demonstrates that CCK-8 activates the brainstem and myenteric neurons through the CCK1 receptor.

The short term satiety peptide cholecystokinin reduces meal size and prolongs intermeal interval

Camostat mesilate (or mesylate) releases endogenous cholecystokinin (CCK) or CCK-58, the only detectable endocrine form of CCK in the rat, and reduces cumulative food intake by activating CCK(1) receptor. However, the literature lacks meal pattern analysis and an appropriate dose-response curve for this peptide. Therefore, the current study determines meal size (MS), intermeal interval (IMI) and satiety ratio (SR) by orogastric gavage of camostat (0, 12.5, 25, 50, 100, 200, 300, 400, 800mg/kg) and compares them to those previously reported by a single dose of CCK-8 (1nmol/kg, i.p), the most utilized form of CCK. We found that camostat (200, 300, 400 and 800mg/kg) and CCK-8 reduced cumulative food intake and the size of the first meal, but only camostat prolonged IMI and increased SR. There was no change in the duration of the first two meals or in rated behaviors such as feeding, grooming, standing and resting in response to camostat and CCK-8, but there was more resting during the IMI in response to camostat. This study provides meal pattern analysis and an appropriate dose-response curve for camostat and CCK-8. Camostat reduces food intake by decreasing MS and prolonging IMI, whereas CCK-8 reduces food intake by reducing only meal size.

The Role of Bombesin and Bombesin-Related Peptides in the Short-term Control of Food Intake

Bombesin (Bn) is a 14-amino acid peptide isolated from the skin of the frog Bombina bombina. The mammalian homologs of this peptide include three forms of gastrin-releasing peptide (GRP): GRP-10, GRP-27, and GRP-29, and a 10-amino acid peptide referred to as neuromedin-B (NMB). These peptides evoke a number of responses, including hyperthermia, bradycardia, inhibition of gastric emptying and inhibition of food intake, by activating one of three G protein-coupled receptors: an NMB-R or BB(1), a GRP-R or BB(2) and an orphan Bn receptor subtype-3 (BRS-3) or BB(3). Bombesin, GRP, and NMB have a role in the short-term control of food intake. These peptides reduce meal size (MS) and they prolong the intermeal interval (IMI), the time between the first and second meals. Studies have shown that the vagus and the splanchnic nerves in the upper gastrointestinal tract, which communicate with the feeding areas of the hindbrain, are necessary for reduction of MS and prolongation of the IMI by Bn, GRP, and NMB. In addition, one-tenth of the intraperitoneal dose of Bn, GRP, and NMB given in either the left gastric artery, which supplies the stomach, or the cranial mesenteric artery, which supplies the intestine, or the femoral vein, also reduces MS and prolongs the IMI. Thus, a potential neurocrine or an endocrine mode of action for these peptides requires further investigation.

Cholecystokinin1 receptors mediate the increase in Fos-like immunoreactivity in the rat myenteric plexus following intestinal oleate infusion

Intestinal infusion of nutrients, such as glucose and oleic acid, increase Fos-like immunoreactivity (Fos-LI) in both the enteric nervous system and neurons of the dorsal vagal complex (DVC) of the hindbrain. To test the hypothesis that increased Fos-LI in enteric neurons and the DVC, following intestinal nutrient infusions is mediated by cholecystokinin(1) receptors (CCK(1)), we counted enteric and DVC neurons that expressed Fos-LI following intestinal infusion of oleate or glucose, with and without pretreatment with the CCK(1) receptor antagonist, lorglumide. Both oleate and glucose infusions increased Fos-LI in the DVC. Oleate also increased Fos-LI in the myenteric and submucosal plexuses of the duodenum and the jejunum, but not the ileum, while glucose only increased Fos-LI in the submucosal plexus of the ileum. The CCK(1) receptor antagonist, lorglumide, abolished Fos-LI in the DVC following infusions of either oleate or glucose. In addition, lorglumide attenuated oleate-induced Fos-LI in the myenteric and submucosal plexuses of the duodenum and jejunum. However, lorglumide failed to attenuate glucose-induced Fos-LI in the submucosal plexus of the ileum. These data confirm previous reports indicating that CCK(1) receptors mediate increased DVC Fos-LI following intestinal infusion of oleate or glucose. CCK(1) receptors also contribute to increased Fos-LI in enteric neurons following intestinal oleate infusion. However, failure of lorglumide to attenuate the increase of Fos-LI in the ileal submucosal plexus following intestinal glucose suggests that some intestinal nutrients trigger Fos-LI induction via CCK(1) receptor-independent pathways.

Cholecystokinin-33 inhibits meal size and prolongs the subsequent intermeal interval.

There are various forms of the satiety gut-brain peptide cholecystokinin (CCK), a short, widely utilized form or CCK-8, and a long, putatively more effective form or CCK-33. The issue of which of these forms is a more effective satiety peptide is not resolved. Here, we compared the satiety responses, including the sizes of the first three meals (MS) and intermeal intervals (IMI) as well as their calculated satiety ratios (SR), evoked by both peptides. CCK-8 and 33 (1, 3 and 5 nmol/kg, i.p) reduced the size of the first meal similarly, only CCK-33 prolonged the first IMI and increased SR and both peptides failed to affect second and third MS and IMI. As such, CCK-33 is a more effective satiety peptide than CCK-8. The current results confirm previous findings which showed that both peptides reduce food intake by inhibiting meal size, whereas only CCK-33 reduces food intake by prolonging the intermeal interval.

Strain differences in myenteric neuron number and CCK1 receptor mRNA expression may account for differences in CCK induced c-Fos activation.

We utilized a diaminobenzidine reaction enhanced with nickel to compare dorsal vagal complex (DVC) and myenteric neuronal Fos-Like immunoreactivity (Fos-LI), in response to sulfated cholecystokinin-8 (CCK-8) (5, 10, 20, 40 microg/kg), among Sprague-Dawley (SD), Standard Long-Evans (SLE), Otsuka Long-Evans Tokushima Fatty (OLETF), and Long-Evans Tokushima Otsuka (LETO) rats. All rat strains but OLETF expressed Fos-LI in response to CCK-8. In addition, SD rats expressed more Fos-LI in the area postrema and myenteric neurons than SLE and LETO rats. To investigate the basis for these differences, we utilized cuprolinic blue staining, which stains neuronal cell bodies, to quantify the number of myenteric neurons, and a reverse transcriptase chain polymerase reaction to measure the gene expression of CCK(1) receptor in the gut. We found that SD rats have significantly more duodenal myenteric neurons than the other strains. In addition, this strain expressed significantly higher levels of the CCK(1) gene in both the duodenum and jejunum than the other strains. In conclusion, SD rats may express more myenteric Fos-LI in response to CCK due to increased numbers of myenteric neurons or more intestinal CCK(1) receptors than the other strains of rats.