John P. Vu

PAC1 deficiency in a murine model induces gastric mucosa hypertrophy and higher basal gastric acid output.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to increase the histamine release from gastric enterochromaffin-like (ECL) cells and promote gastric acid secretion in rats. In contrast, in mice, PACAP has been demonstrated to induce a decrease of gastric acid secretion, an effect presumably due to somatostatin release. To more clearly define the role of PACAP in the regulation of gastric acid output, a knockout mouse model for the PACAP-specific receptor PAC1 was applied in this study. Measurements of the basal and stimulated gastric acid secretion and morphological studies on the gastric mucosa were performed in both wild-type and PAC1-deficient mice. Compared with the wild-type mice, the PAC1-deficient mice showed a nearly threefold higher basal gastric acid output, increased gastric mucosa thickness and glands height, and proportional increases in parietal and total cell counts in the gastric mucosa. The PAC1-deficient mice also showed a trend of increased plasma gastrin levels and gastrin gene expression in the gastric mucosa. This study indicates that the expression of PAC1 is clearly important for maintaining the homeostasis of gastric acid secretion. Loss of PACAP receptor during development may lead to a compensatory mechanism regulating gastric acid secretion.

Ghrelin in neuroendocrine tumors.

Ghrelin is a 28 amino acid peptide, primarily produced by the oxyntic mucosa X/A like neuroendocrine cells in the stomach. It is also found in the small intestine, hypothalamus, pituitary gland, pancreas, heart, adipose tissue, and immune system. In gastrointestinal neuroendocrine tumors (NETs) ghrelin release has been well documented. Ghrelin is a brain-gut circuit peptide with an important role in the physiological regulation of appetite, response to hunger and starvation, metabolic and endocrine functions as energy expenditure, gastric motility and acid secretion, insulin secretion and glucose homeostasis, as well as in the potential connection to the central nervous system. Recently, there has been a significant interest in the biological effects of ghrelin in NETs. In this article, we present a comprehensive review of ghrelins expression and a brief summary of ghrelins physiological role in NETs patients with carcinoids, type A chronic atrophic gastritis (CAG), with or without MEN-1, and with and without liver metastases. We hope, with the research reviewed here, to offer compelling evidence of the potential significance of ghrelin in NETs, as well as to provide a useful guide to the future work in this area.

PACAP intraperitoneal treatment suppresses appetite and food intake via PAC1 receptor in mice by inhibiting ghrelin and increasing GLP-1 and leptin.

Pituitary adenylate cyclase-activating peptide (PACAP) is expressed within the gastroenteric system, where it has profound physiological effects. PACAP was shown to regulate food intake and thermogenesis centrally; however, PACAP peripheral regulation of appetite and feeding behavior is unknown. Therefore, we studied PACAPs effect on appetite and food intake control by analyzing feeding behavior and metabolic hormones in PAC1-deficient (PAC1-/-) and age-matched wild-type (WT) mice intraperitoneally injected with PACAP1-38 or PACAP1-27 before the dark phase of feeding. Food intake and feeding behavior were analyzed using the BioDAQ system. Active ghrelin, glucagon-like peptide-1 (GLP-1), leptin, peptide YY, pancreatic polypeptide, and insulin were measured following PACAP1-38 administration in fasted WT mice. PACAP1-38/PACAP1-27 injected into WT mice significantly decreased in a dose-dependent manner cumulative food intake and reduced bout and meal feeding parameters. Conversely, PACAP1-38 injected into PAC1-/- mice failed to significantly change food intake. Importantly, PACAP1-38 reduced plasma levels of active ghrelin compared with vehicle in WT mice. In PAC1-/- mice, fasting levels of active ghrelin, GLP-1, insulin, and leptin and postprandial levels of active ghrelin and insulin were significantly altered compared with levels in WT mice. Therefore, PAC1 is a novel regulator of appetite/satiety. PACAP1-38/PACAP1-27 significantly reduced appetite and food intake through PAC1. In PAC1-/- mice, the regulation of anorexigenic/orexigenic hormones was abolished, whereas active ghrelin remained elevated even postprandially. PACAP significantly reduced active ghrelin in fasting conditions. These results establish a role for PACAP via PAC1 in the peripheral regulation of appetite/satiety and suggest future studies to explore a therapeutic use of PACAP or PAC1 agonists for obesity treatment.

Mo1881 Hypergastrinemia Associated With Gastrointestinal Dysmotility Is Mediated by Vasoactive Intestinal Polypeptide (VIP)

G A A b st ra ct s potencies (PLC activation) for BRS-3, GRPR and NMBR of human, rat or mouse on native and transfected cells. Results: GRP and NMB had very low affinity (IC50 >3000 nM) for r,m,hBRS-3 receptors, but high affinity and selectivity for their own receptors (GRPR, NMBR) in all species. Peptide #1 had high affinity for all three human BnRs, and r,mNMBR/GRPR. Because of a lack of a radioligand for r,mBRS-3, their affinities could not be assessed by binding studies, only by potency of cell activation. In human BRS-3 receptors, the relative affinities of the chiral diazepines was 9G (IC50: 70 nM) > 9D (IC50: 121 nM) > 9F (IC50: 495 nM) > 3F (IC50: 908 nM); each was selective for BRS-3, and each did not interact with hGRPR, hNMBR even at concentrations up to 3000 nM, nor did they interact with r,mGRPR, NMBR. For stimulating PLC activity, in hBRS-3 each of the four chiral diazepine analogs was fully efficacious and their relative potencies were: peptide #1 (EC50: 4 nM) > 9G (EC50: 9 nM) > 9D (EC50: 9.4 nM) > 9F (EC50: 39 nM) > 3F (EC50: 48 nM) >>GRP, NMB (EC50 >3000 nM). Comparison of affinities and potencies demonstrated the different diazepine analogs had variable receptor sparseness wirh IC50/EC50 ratios of: 7.7 (9G), 12.8 (9D), 12.6 (9F), 18.9 (3F). None of the four chiral diazepine analogs activated r,m,hGRPR, NMBR even at concentrations of 3000 nM. Conclusions: This study demonstrates the recently described chiral diazepines analogs (9G, 9D, 9F, 3F) are fully efficacious agonists for the BRS-3 receptor in human and rhodent cells. Furthermore, they are highly selective for this receptor, as well as having high potency, and should prove useful for exploring its role in GI physiological and pathological processes.

Mo1986 Vasoactive Intestinal Peptide (VIP) Plays a Role in the Regulation of Orexigenic and Anorexigenic Hormones and Body Fat and Lean Mass Composition

Aim: To create HEK293 cells and murine ES cell lines that synthesize and release either GIP or the specific GIPR antagonist GIP(5-42). Methods: Using PCR, chimeric genes, consisting of the mouse growth hormone leader (mGH), the furin cleavage site, and either GIP or GIP(5-42), were generated and cloned into a lentiviral vector downstream of a constitutive promoter. After the generation of lentiviral pseudoparticles containing the chimeric genes, mGH-GIP and mGH-GIP(5-42), HEK 293 and ES cells were transduced with ~108 pseudoparticles. Pools of cells containing the co-expressed fluorescent marker ZsGreen were isolated using flow cytometry before supernates from cultured cells were collected, and a specific ELISA was employed to quantify GIP immunoreactivity. To assess GIP bioactivity in supernates, a specific bioassay consisting of reporter cells expressing GIPR and containing the LacZ gene under the control of a cAMP-responsive promoter was used. To demonstrate antagonist activity, peptides in supernates from HEK 293 cells containing mGH-GIP(5-42) were concentrated 50-fold using a C-18 reverse phase column before being mixed with 1010 M GIP and added to reporter cells. Results: HEK 293 cells containing mGH-GIP and mGH-GIP(5-42) expressed 8x10-4 and 10-4 pg/cell/h, respectively, while ES cells containing mGH-GIP and mGH-GIP(5-42) expressed 10-4 and 6x10-3 pg/cell/h, respectively. Supernates from HEK 293 and ES cells containing mGH-GIP were both shown to induce LacZ expression, demonstrating the presence of bioactive GIP. As expected, supernates from either HEK 293 cells or ES cells containing mGH-GIP(5-42) did not induce LacZ expression. However, after concentration, supernates from HEK 293 cells containing mGH-GIP(5-42) inhibited LacZ expression induced by 10-10 M GIP, indicating the presence of antagonist activity. Summary and Conclusion: The results of these studies demonstrate the successful engineering of HEK 293 and ES cells to express bioactive GIP and GIP(5-42). Although early in its development, engraftment of tissue derived fromGIP(5-42) expressing stem cells may provide a method for treating obesity.

Mo1110 VIP Antagonism Has an Anti-Inflammatory Effect in Dextran Sulfate Sodium (DSS)-Induced Colitis

Introduction: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with a poorly understood pathogenesis. Vasoactive Intestinal Peptide (VIP) has been shown to play a potent anti-inflammatory effect In Vitro, by inhibiting the release of Th1 pro-inflammatory cytokines from T cells and macrophages. Thus, our expectation is that the use of a VIP antagonist or deletion of the VIP gene in mice would lead to an increase in inflammation. To investigate this hypothesis further In Vivo, we have utilized the well characterized Dextran Sulfate Sodium (DSS) colitis model, to study the development of colonic inflammation in wild type (WT) mice, with or without the administration of VIP partial antagonist (VIPHybrid) and in VIP-/deficient mice. Methods: Colitis was induced in 8 adult (WT), and 8 adult VIP -/deficient, C57BL/6 mice with 2.5% DSS given orally for 5 days. Intraperitoneal (ip) injections of 2.3 ug (100uM) VIPHybrid (Phoenix) or vehicle were administered daily to the WT mice. Measurements of water intake, body weight, observations of activity and stool appearance were made at baseline and daily thereafter for eleven consecutive days. On the 11th day the animals were sacrificed and the intact colon from each mouse was dissected, examined macroscopically, the weight and length were measured and their ratio was scored in a blinded fashion. The myeloperoxidase (MPO) assay was performed on samples from the proximal and distal colon for each mouse. Results: WT mice, treated with vehicle showed the greatest loss in body weight (11.5%) compared to the mice treated with VIPHybrid (8.9%, p<0.05) or VIP -/(1.4%, p<0.001). The colonic length to weight ratio was significantly lower in the VIPHybrid treated group (2.9, p<0.05) and VIP -/group (3.0, p<0.01) compared to the vehicle treated group (3.7). MPO assay and histological analysis on H&E stained cut tissue slides performed on proximal and distal colonic samples confirmed the lower values of the inflammatory parameters observed in VIP-/and in VIPHybrid treated WT mice groups. Conclusions: DSS-treated VIP deficient mice exhibited significantly lower levels of inflammatory parameters, compared to WT mice. WT mice treated with VIPHybrid also demonstrated lower inflammatory parameters than untreated WT mice. These results demonstrate that VIP antagonism as shown by both using a VIP receptor antagonist or in VIP deficient mice leads to a reduction in colonic inflammation. Thus, VIP antagonists such as VIPHybrid may provide to be a useful strategy to reduce colonic inflammation in conditions such as inflammatory bowel disease and deserve further investigation.