Deoxynivalenol (Vomitoxin)-Induced Anorexia Is Induced by the Release of Intestinal Hormones in Mice

Deoxynivalenol (DON), also known as vomitoxin, is a mycotoxin that can cause antifeeding and vomiting in animals. However, the mechanism of DON inducing anorexia is complicated. Studies have shown that intestinal hormones play a significant part in the anorexia caused by DON. We adopted the “modeling of acute antifeeding in mice” as the basic experimental model, and used two methods of gavage and intraperitoneal injection to explore the effect of intestinal hormones on the antifeedant response induced by DON in mice. We found that 1 and 2.5 mg/kg·bw of DON can acutely induce anorexia and increase the plasma intestinal hormones CCK, PYY, GIP, and GLP-1 in mice within 3 h. Direct injection of exogenous intestinal hormones CCK, PYY, GIP, and GLP-1 can trigger anorexia behavior in mice. Furthermore, the PYY receptor antagonist JNJ-31020028, GLP-1 receptor antagonist Exendin(9-39), CCK receptor antagonist Proglumide, GIP receptor antagonist GIP(3-30)NH2 attenuated both intestinal hormone and DON-induced anorectic responses. These results indicate that intestinal hormones play a critical role in the anorexia response induced by DON.

4119 Cholecystokinin (CCK) Receptor Antagonist Reverses Nonalcoholic Steatohepatitis (NASH) by Reducing Hepatic Macrophages and Inflammatory Cytokines

OBJECTIVES/GOALS: NASH increases the risk of cirrhosis and liver cancer. High-fat diets increase CCK levels and CCK receptors have been identified on fibroblasts and immune cells. We hypothesized that CCK receptor blockade could prevent NASH by altering the hepatic microenvironment and macrophage activation. METHODS/STUDY POPULATION: Female mice were fed a Choline Deficient Ethionine supplemented (CDE) saturated fat diet or control high-fat diet for 18 weeks. Mice in each group were treated with a CCK receptor antagonist, proglumide (0.1 mg/ml) in the drinking water or regular water. Resected livers were stained for H&E for features of NASH and F4/80 for macrophages analysis. Liver RNA was evaluated for the expression of cytokines and chemokines using an 84-gene Profiler array (Qiagen). Oxidative stress was analyzed by qRT-PCR for heat shock proteins (HSPs) 27, 60, 70 and 90 and for glutathione by a fluorometric assay. Differences in CDE fed and CDE/proglumide-treated mouse livers were evaluated. RESULTS/ANTICIPATED RESULTS: Livers from mice on the CDE diet displayed histologic features of NASH that were prevented by proglumide. Cytokines and chemokines expression, especially CCL20 and CCL2, were increased in the CDE fed mice and these levels were reduced greater than 20-fold with proglumide. Infiltration of F4/80+ macrophages was markedly increased in the CDE livers and these were reduced by > 50% (p < 0.0001) with proglumide. RNA expression of HSP70 (p = 0.006) and HSP27 (p = 0.011) were reduced with proglumide. Hepatic glutathione concentration more than doubled in the CDE/proglumide treated mice compared to CDE mice. CCK-B receptor expression increased in the CDE-fed mouse livers compared to controls. DISCUSSION/SIGNIFICANCE OF IMPACT: CCK receptor blockade decreases NASH by reducing hepatic macrophages, oxidative stress, and blocking inflammatory cytokines and chemokines. This data supports our novel hypothesis that CCK receptors play a role in NASH and proglumide may provide an innovative treatment for this condition.

Dietary fat stimulates pancreatic cancer growth and promotes fibrosis of the tumor microenvironment through the cholecystokinin receptor

The gastrointestinal peptide cholecystokinin (CCK) is released from the duodenum in response to dietary fat to aid in digestion, and plasma CCK levels are elevated with the consumption of high-fat diets. CCK is also a trophic peptide for the pancreas and has also been shown to stimulate growth of pancreatic cancer. In the current investigation, we studied the influence of a diet high in saturated fat on the growth of pancreatic cancer in syngeneic murine models before the mice became obese to exclude the confounding factors associated with obesity. The high-fat diet significantly increased growth and metastasis of pancreatic cancer compared with the control diet, and the stimulatory effect was blocked by the CCK-receptor antagonist proglumide. We then selectively knocked out the CCK receptor on the pancreatic cancer cells using clustered regularly interspaced short palindromic repeats technology and showed that without CCK-receptors, dietary fat was unable to stimulate cancer growth. We next demonstrated that dietary fat failed to influence pancreatic cancer xenograft growth in genetically engineered CCK peptide knockout mice. The tumor-associated fibrosis that is so prevalent in the pancreatic cancer microenvironment was significantly decreased with CCK-receptor antagonist therapy because fibroblasts also have CCK receptors. The CCK-receptor antagonist proglumide also altered tumor metalloprotease expression and increased tumor suppressor genes by a PCR array. Our studies confirm that a diet high in saturated fat promotes growth of pancreatic cancer and the action is mediated by the CCK-receptor pathway. NEW & NOTEWORTHY Diets high in long-chain saturated fats promote growth of pancreatic cancer independent of obesity. The mechanism through which dietary fat promotes cancer is mediated through the cholecystokinin (CCK) receptor pathway. Therapy with a CCK-receptor antagonist altered the tumor microenvironment by reducing fibrosis, increasing cluster of differentiation 8+ lymphocytes, increasing tumor suppressor genes, and thus decreasing metastases. Use of CCK-receptor antagonist therapy with standard chemotherapy for pancreatic cancer may improve response by altering the tumor microenvironment.

Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu7.39, whereas the role of this residue was less clear for chemically distinct agonists.

The effect of cholecystokinin peptides on ovine duodeno-jejunal slow waves with and without pretreatment with proglumide

Cholecystokinin exerts a composite influence on gastrointestinal motility but little is known about its effect on small-intestinal slow waves. Thus, six rams were implanted with four bipolar serosal electrodes onto the duodeno-jejunal wall. In the course of chronic experiments the myoelectric activity was continuously recorded in the non-fasted animals. After recording of the full normal migrating myoelectric complex (MMC), 0.15 M NaCl or CCK peptides were injected intravenously during various phases of the next MMC cycle. Five mℓ of saline was injected over 30 s during phases 1, 2a, or 2b of the MMC. Cerulein was administered at doses of 1 (over 30 s), 10 (over 30 or 60 s), or 100 ng/kg (over 30, 60, 120 or 300 s) and cholecystokinin octapeptide (CCK-OP) at doses 20 times higher. CCK peptides were applied during early or late phase 1 of the MMC and during phases 2a and 2b of the MMC. In the course of additional experiments, saline and hormone administration was directly preceded by infusion of proglumide, an unspecific CCK receptor antagonist, at a dose of 10 mg/kg. The myoelectric recordings were continued until the arrival of a subsequent regular phase 3 of theMMC.In the duodenal bulb, slow waves were occasionally observed. In the duodenum the slow-wave frequency oscillated between 20 and 24 cpm and in the jejunum between 19 and 22 cpm before or after CCK peptides and proglumide. In the duodenum the slow-wave amplitude increased significantly after all doses of cerulein injected during phase 2b of the MMC. After administration of CCK-OP changes in duodenal slow-wave amplitude were not significant but exhibited a tendency similar to those after cerulein. In the jejunum, injection of cerulein and CCK-OP during phase 2 of the MMC increased the slow-wave amplitude significantly and the duration of these changes was longer than in the duodenum. After infusion of proglumide, administration of cerulein at the low dose over 30 s and at the high dose over 300 s in the course of late phase 1 and phases 2a and 2b of the MMC, significantly increased the duodenal slow-wave amplitude. Cerulein injection during phase 2b of the MMC at the high dose over 30 and 60 s, preceded by proglumide infusion, significantly inhibited the duodenal slow-wave amplitude. In the jejunum these changes were even more pronounced and their duration was much longer. It is concluded that CCK peptides affect slow-wave amplitude in the duodeno-jejunum in non-fasted sheep. This effect is stronger in the jejunum and is altered but not abolished by pretreatment with proglumide. Cerulein evokes more pronounced alterations in the slow-wave amplitude than CCK-OP in conscious sheep.

Feeding motivation in swine: Relation with insulin, glucose and free fatty acids in portal and jugular blood, and involvement of cholecystokinin

The control of feeding motivation was studied in eight castrated male pigs (92.9 ± 1.5 kg) surgically fitted with jugular and portal vein catheters. The first part of the experiment was set up as a cross-over design. Two weeks after surgery, four pigs were fasted for 24 h (FAST) while the remaining four were fed ad libitum (CTL). Three days later, the same procedure was repeated with the treatments reversed. Blood samples were collected every 4 h during the first 16 h of these two 24-h periods. After each 24-h period, pigs were subjected to a feed motivation test during which blood samples were obtained every 5 min for 60 min for CTL pigs and until 30 min after cessation of eating in FAST pigs. Three days later, the second part of the experiment was conducted. Five of the eight pigs were fasted for another 24 h, injected i.v. with 70 μg kg–1 BW of a cholecystokinin (CCK) receptor antagonist (MK-329), and subsequently subjected to a feed motivation test. Insulin, glucose and FFA concentrations were greater (P < 0.001) in portal than jugular blood. Fasting decreased insulin concentrations in both portal and jugular blood, decreased glucose in portal blood only, and increased FFA in jugular blood only. The number of pushes (P < 0.05), duration of eating (P < 0.001) and amount of feed eaten during the feed motivation test (P < 0.01) were all increased by fasting, and were further increased with MK-329 injection. Results indicate that CCK is involved in the control of satiety in pigs and that concentrations of insulin, glucose and FFA in the jugular vein may not always be representative of concentrations in the portal vein. Key words: Swine, insulin, glucose, cholecystokinin, feeding motivation, fast

Effects of M1 and CCK antagonists on latency of pancreatic amylase response to intestinal stimulants

In six conscious dogs with gastric and duodenal cannulas, secretin (164 pmol · kg−1 · h−1 iv) was given to provide a flow of pancreatic juice of ∼1 drop/s. Amylase activity was measured in each drop before and after rapid intravenous injection of caerulein (7.4 pmol/kg) or intraduodenal injection of l-tryptophan (1 mmol), sodium oleate (3 mmol), and HCl (3 mmol). All experiments were repeated in the presence of the M1 receptor antagonist telenzepine (81 nmol · kg−1 · h− iv) and the cholecystokinin (CCK) receptor antagonist L-364718 (0.1 mg/kg iv). Latency of amylase response (time between injection of stimulant and sustained increase in amylase activity greater than mean + 3 SD of prestimulatory activity) to tryptophan (17 ± 7 s; n = 6) and oleate (16 ± 5 s) was significantly ( P < 0.05) shorter than to caerulein (28 ± 4 s) and HCl (120 ± 47 s). Telenzepine significantly increased the latency of amylase response to tryptophan and oleate by >10-fold but not the latency to caerulein or HCl. L-364718 abolished the amylase response to all stimulants. These findings indicate that the early amylase response to intraduodenal tryptophan and oleate is mediated by a neural enteropancreatic reflex ending on M1 receptors rather than by hormone release. However, the activation of (possibly vagal) CCK receptors is essential to run the reflex. The early amylase response to intraduodenal HCl is probably mediated by the release of CCK into the blood circulation.