Screening for positive allosteric modulators of cholecystokinin type 1 receptor potentially useful for management of obesity.

Obesity has become a prevailing health burden globally and particularly in the US. It is associated with many health problems, including cardiovascular disease, diabetes and poorer mental health. Hence, there is a high demand to find safe and effective therapeutics for sustainable weight loss. Cholecystokinin (CCK) has been implicated as one of the first gastrointestinal hormones to reduce overeating and suppress appetite by activating the type 1 cholecystokinin receptor (CCK1R). Several drug development campaigns have focused on finding CCK1R-specific agonists, which showed promising efficacy for reducing meal size and weight, but fell short on FDA approval, likely due to side effects associated with potent, long-lasting activation of CCK1Rs. Positive allosteric modulators (PAMs) without inherent agonist activity have been proposed to overcome the shortcomings of traditional, orthosteric agonists and restore CCK1R signaling in failing physiologic systems. However, drug discovery campaigns searching for such novel acting CCK1R agents remain limited. Here we report a high-throughput screening effort and the establishment of a testing funnel, which led to the identification of novel CCK1R modulators. We utilized IP-One accumulation to develop robust functional equilibrium assays tailored to either detect PAMs, agonists or non-specific activators. In addition, we established the CCK1R multiplex PAM assay as a novel method to evaluate functional selectivity capable of recording CCK1R-induced cAMP accumulation and ß-arrestin recruitment in the same well. This selection and arrangement of methods enabled the discovery of three scaffolds, which we characterized and validated in an array of functional and binding assays. We found two hits incorporating a tetracyclic scaffold that significantly enhanced CCK signaling at CCK1Rs without intrinsically activating CCK1Rs in an overexpressing system. Our results demonstrate that a well-thought-out testing funnel can identify small molecules with a distinct pharmacological profile and provides an important milestone for the development of novel potential treatments of obesity.

Roles of Cholecystokinin in the Nutritional Continuum. Physiology and Potential Therapeutics.

Cholecystokinin is a gastrointestinal peptide hormone with important roles in metabolic physiology and the maintenance of normal nutritional status, as well as potential roles in the prevention and management of obesity, currently one of the dominant causes of direct or indirect morbidity and mortality. In this review, we discuss the roles of this hormone and its receptors in maintaining nutritional homeostasis, with a particular focus on appetite control. Targeting this action led to the development of full agonists of the type 1 cholecystokinin receptor that have so far failed in clinical trials for obesity. The possible reasons for clinical failure are discussed, along with alternative pharmacologic strategies to target this receptor for prevention and management of obesity, including development of biased agonists and allosteric modulators. Cellular cholesterol is a natural modulator of the type 1 cholecystokinin receptor, with elevated levels disrupting normal stimulus-activity coupling. The molecular basis for this is discussed, along with strategies to overcome this challenge with a corrective positive allosteric modulator. There remains substantial scope for development of drugs to target the type 1 cholecystokinin receptor with these new pharmacologic strategies and such drugs may provide new approaches for treatment of obesity.

Activation of multiple receptors stimulates extracellular vesicle release from trophoblast cells.

Reports of the stimulated release of extracellular vesicles (EVs) are few, and the mechanisms incompletely understood. To our knowledge, the possibility that the activation of any one of the multitudes of G-protein-coupled receptors (GPCRs) expressed by a single cell-type might increase EV release has not been explored. Recently, we identified the expression of cholecystokinin (CCK), gastrin, gastrin/cholecystokinin types A and/or B receptors (CCKAR and/or -BR), and the bitter taste receptor, TAS2R14 in the human and mouse placenta. specifically, trophoblast. These GPCR(s) were also expressed in four different human trophoblast cell lines. The current objective was to employ two of these cell lines-JAR choriocarcinoma cells and HTR-8/SVneo cells derived from first-trimester human villous trophoblast-to investigate whether CCK, TAS2R14 agonists, and other GPCR ligands would each augment EV release. EVs were isolated from the cell-culture medium by filtration and ultracentrifugation. The preparations were enriched in small EVs (<200 nm) as determined by syntenin western blot before and after sucrose gradient purification, phycoerythrin (PE)-ADAM10 antibody labeling, and electron microscopy. Activation of TAS2R14, CCKBR, cholinergic muscarinic 1 & 3, and angiotensin II receptors, each increased EV release by 4.91-, 2.79-, 1.87-, and 3.11-fold, respectively (all p < .05 versus vehicle controls), without significantly changing EV diameter. A progressive increase of EV concentration in conditioned medium was observed over 24 hr consistent with the release of preformed EVs and de novo biogenesis. Compared to receptor-mediated stimulation, EV release by the calcium ionophore, A23187, was less robust (1.63-fold, p = .08). Diphenhydramine, a TAS2R14 agonist, enhanced EV release in JAR cells at a concentration 10-fold below that required to increase intracellular calcium. CCK activation of HTR-8/SVneo cells, which did not raise intracellular calcium, increased EV release by 2.06-fold (p < .05). Taken together, these results suggested that other signaling pathways may underlie receptor-stimulated EV release besides, or in addition to, calcium. To our knowledge, the finding that the activation of multiple GPCRs can stimulate EV release from a single cell-type is unprecedented and engenders a novel thesis that each receptor may orchestrate intercellular communication through the release of EVs containing a subset of unique cargo, thus mobilizing a specific integrated physiological response by a network of neighboring and distant cells.

Cholecystokinin-1 receptor agonist induced pathological findings in the exocrine pancreas of non-human primates.

BACKGROUND AND AIMS: Cholecystokinin (CCK) may potentially be used to treat obesity. However, it is well-known to induce acute pancreatitis and pancreas neoplasia in rodents, but not in primates. Here we report the nonclinical safety profile of a long-acting CCK-1 receptor (CCK-1R) agonist, NN9056, in rats and monkeys to support a First-in-Man clinical trial with NN9056. METHODS: Thirteen-week toxicological studies were conducted in rats and non-human primates followed by histopathological evaluation of affected tissues. NN9056 was characterised in vitro, and CCK-1R expression was assessed by in situ hybridization in cynomolgus monkey and human pancreas tissues. RESULTS: Affinity and potency of NN9056 was comparable to native sulphated CCK-8 (CCK-8) across species on the CCK-1R while it had no effect on the CCK-2 receptor (CCK-2R). In situ hybridization demonstrated abundant expression of CCK-1Rs in the exocrine pancreas of the rat. In contrast, it was only discreetly expressed on pancreatic acinar cells in the periphery of scattered lobules in monkeys. A similar expression pattern was observed in human pancreas. 13-weeks daily dosing with NN9056 produced the expected pancreatic pathological findings in rats. In monkeys, NN9056 increased pancreas weight and induced histopathological changes despite the low expression level of CCK-1Rs. CONCLUSION: Surprisingly, chronic CCK-1R activation constitutes a risk for pancreatitis and trophic actions on the exocrine pancreas in monkeys. Since similar CCK-1R expression patterns were found in pancreas of monkeys and humans this risk is likely translatable to humans and clinical development of NN9056 was therefore halted.

CCK-1 and CCK-2 receptor agonism do not stimulate GLP-1 and neurotensin secretion in the isolated perfused rat small intestine or GLP-1 and PYY secretion in the rat colon.

Gastrin and cholecystokinin (CCK) are hormones released from endocrine cells in the antral stomach (gastrin), the duodenum, and the jejunum (CCK). Recent reports, based on secretion experiments in an enteroendocrine cell line (NCI-H716) and gastrin receptor expression in proglucagon-expressing cells from the rat colon, suggested that gastrin could be a regulator of glucagon-like peptide-1 (GLP-1) secretion. To investigate these findings, we studied the acute effects of CCK-8 (a CCK1/CCK2 (gastrin) receptor agonist) and gastrin-17 (a CCK2(gastrin) receptor agonist) in robust ex vivo models: the isolated perfused rat small intestine and the isolated perfused rat colon. Small intestines from Wistar rats (n = 6), were perfused intraarterially over 80 min. During the perfusion, CCK (1 nmol/L) and gastrin (1 nmol/L) were infused over 10-min periods separated by washout/baseline periods. Colons from Wistar rats (n = 6) were perfused intraarterially over 100 min. During the perfusion, CCK (1 nmol/L), vasoactive intestinal peptide (VIP) (10 nmol/L), and glucose-dependent insulinotropic polypeptide (GIP) (1 nmol/L) were infused over 10-min periods separated by washout/baseline periods. In the perfused rat small intestines neither CCK nor gastrin stimulated the release of GLP-1 or neurotensin. In the perfused rat colon, neither CCK or VIP stimulated GLP-1 or peptide YY (PYY) release, but GIP stimulated both GLP-1 and PYY release. In both sets of experiments, bombesin, a gastrin-releasing peptide analog, served as a positive control. Our findings do not support the suggestion that gastrin or CCK participate in the acute regulation of intestinal GLP-1 secretion, but that GIP may play a role in the regulation of hormone secretion from the colon.

Cholecystokinin Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis.

BACKGROUND AND AIMS: Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. METHODS: Two murine models were used to determine whether CCK receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18 weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. RESULTS: Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFßR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. CONCLUSION: New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.

Cholecystokinin, gastrin, cholecystokinin/gastrin receptors, and bitter taste receptor TAS2R14: trophoblast expression and signaling.

We investigated expression of cholecystokinin (CCK) in humans and mice, and the bitter taste receptor TAS2R14 in the human placenta. Because CCK and gastrin activate the CCKBR receptor, we also explored placental gastrin expression. Finally, we investigated calcium signaling by CCK and TAS2R14. By RT-PCR, we found CCK/Cck and GAST/Gast mRNA expression in both normal human and mouse placentas, as well as in human trophoblast cell lines (TCL). Although both Cckar and -br mRNA were expressed in the mouse placenta, only CCKBR mRNA was detected in the human placenta and TCL. mRNA expression for TAS2R14 was also observed in the human placenta and TCL. Using immunohistochemistry, CCK protein was localized to the syncytiotrophoblast (ST) and extravillous trophoblast (EVT) in the human term placenta, and to trophoblast glycogen cells in mouse and human placentas. Gastrin and TAS2R14 proteins were also observed in ST and EVT of the human placenta. Both sulfated and nonsulfated CCK elicited a comparable rise in intracellular calcium in TCL, consistent with CCKBR expression. Three TAS2R14 agonists, flufenamic acid, chlorhexidine, and diphenhydramine, also evoked rises in intracellular calcium in TCL. These results establish CCK, gastrin, and their receptor(s) in both human and mouse placentas, and TAS2R14 in the human placenta. Both CCK and TAS2R14 agonists increased intracellular calcium in human TCL. Although the roles of these ligands and receptors, and their potential cross talk in normal and pathological placentas, are currently unknown, this study opens new avenues for placental research.

Structure-Activity Relationships and Characterization of Highly Selective, Long-Acting, Peptide-Based Cholecystokinin 1 Receptor Agonists.

A group of peptide-based, long-acting, stable, highly selective cholecystokinin 1 receptor (CCK-1R) agonists with the potential to treat obesity has been identified and characterized, based on systematic investigation of synthetic CCK-8 analogues with N-terminal linkage to fatty acids. Sulfated Tyr in such compounds was stable in neutral buffer. CCK-1R selectivity was achieved mostly by introducing d- N-methyl-Asp instead of Asp at the penultimate position of CCK-8. Our compound 9 (NN9056) showed similar in vitro CCK-1R potency and CCK-1R affinity as CCK-8, very high selectivity for CCK-1R over the cholecystokinin 2 receptor (CCK-2R), strong reduction of food intake in lean pigs for up to 48 h after one subcutaneous injection without adverse effects, a plasma half-life of 113 h in minipigs after intravenous injection, and acceptable chemical stability in a neutral liquid formulation. In addition, we found a highly selective CCK-2R agonist by replacing Gly in a CCK-8 derivative with Glu.

Molecular Basis of Action of a Small-Molecule Positive Allosteric Modulator Agonist at the Type 1 Cholecystokinin Holoreceptor.

Allosteric modulation of receptors provides mechanistic safety while effectively achieving biologic endpoints otherwise difficult or impossible to obtain by other means. The theoretical case has been made for the development of a positive allosteric modulator (PAM) of the type 1 cholecystokinin receptor (CCK1R) having minimal intrinsic agonist activity to enhance meal-induced satiety for the treatment of obesity, while reducing the risk of side effects and/or toxicity. Unfortunately, such a drug does not currently exist. In this work, we have identified a PAM agonist of the CCK1R, SR146131, and determined its putative binding mode and receptor activation mechanism by combining molecular modeling, chimeric CCK1R/CCK2R constructs, and site-directed mutagenesis. We probed the structure-activity relationship of analogs of SR146131 for impact on agonism versus cooperativity of the analogs. This identified structural features that might be responsible for binding affinity and potency while retaining PAM activity. SR146131 and several of its analogs were docked into the receptor structure, which had the natural endogenous peptide agonist, cholecystokinin, already in the bound state (by docking), providing a refined structural model of the intact CCK1R holoreceptor. Both SR146131 and its analogs exhibited unique probe-dependent cooperativity with orthosteric peptide agonists and were simultaneously accommodated in this model, consistent with the derived structure-activity relationships. This provides improved understanding of the molecular basis for CCK1R-directed drug development.

Involvement of Microflora Metabolites in Up-Regulation of Electrical Activity in Rat Small Intestine.

The study examined implication of tributyrin, a metabolic precursor in small intestinal microflora, in stimulation of electrical activity of duodenum and jejunum. Enteral administration of tributyrin enhanced electrical activity of both examined structures in small intestine with elimination of the rest periods. The stimulatory effect was manifested in a prolongation of the periods of irregular activity. The up-regulating effects of tributyrin on electrical activity in upper segments of small intestine are mediated via the cholecystokinin receptors being also associated with activation of cholinergic and blockade of nitrinergic signal transduction pathways.

Impaired intestinal cholecystokinin secretion, a fascinating but overlooked link between coeliac disease and cholesterol gallstone disease.

BACKGROUND: Coeliac disease is a chronic, small intestinal, immune-mediated enteropathy caused by a permanent intolerance to dietary gluten in genetically predisposed individuals. Clinical studies have found that intestinal cholecystokinin secretion and gallbladder emptying in response to a fatty meal are impaired before coeliac patients start the gluten-free diet (GFD). DESIGN: However, it was never really appreciated whether coeliac disease is associated with gallstones because there were very few studies investigating the mechanism underlying the impact of coeliac disease on the pathogenesis of gallstones. RESULTS: We summarize recent progress on the relationship between coeliac disease and gallstones and propose that coeliac disease is an important risk factor for gallstone formation because defective intestinal cholecystokinin secretion markedly increases susceptibility to cholesterol gallstones via a mechanism involving dysmotility of both the gallbladder and the small intestine. Because GFD can significantly improve the coeliac enteropathy, early diagnosis and therapy in coeliac patients is crucial for preventing the long-term impact of cholecystokinin deficiency on the biliary and intestinal consequences. When gluten is reintroduced, clinical and histologic relapse often occurs in coeliac patients. Moreover, some of the coeliac patients do not respond well to GFD. CONCLUSIONS: It is imperative to routinely examine by ultrasonography whether gallbladder motility function is preserved in coeliac patients and monitor whether biliary sludge (a precursor of gallstones) appears in the gallbladder, regardless of whether they are under the GFD programme. To prevent gallstones in coeliac patients, it is urgently needed to investigate the prevalence and pathogenesis of gallstones in these patients.

A whole animal chemical screen approach to identify modifiers of intestinal neutrophilic inflammation.

By performing two high-content small molecule screens on dextran sodium sulfate- and trinitrobenzene sulfonic acid-induced zebrafish enterocolitis models of inflammatory bowel disease, we have identified novel anti-inflammatory drugs from the John Hopkins Clinical Compound Library that suppress neutrophilic inflammation. Live imaging of neutrophil distribution was used to assess the level of acute inflammation and concurrently screen for off-target drug effects. Supporting the validity of our screening strategy, most of the anti-inflammatory drug hits were known antibiotics or anti-inflammatory agents. Novel hits included cholecystokinin (CCK) and dopamine receptor agonists. Using a pharmacological approach, we show that while CCK and dopamine receptor agonists alleviate enterocolitis-associated inflammation, receptor antagonists exacerbate inflammation in zebrafish. This work highlights the utility of small molecule screening in zebrafish enterocolitis models as a tool to identify novel bioactive molecules capable of modulating acute inflammation.

Copeptin - A potential endocrine surrogate marker of CCK-4-induced panic symptoms?

Intravenous cholecystokinin-tetrapeptide (CCK-4) administration reliably and dose-dependently provokes panic anxiety in man, accompanied by adrenocorticotropic hormone (ACTH) and cortisol release. Preclinical findings suggest that behavioral and endocrine effects of CCK-4 are mediated via corticotropin-releasing hormone (CRH) release. Anxiogenic stimulation of the central CCK-receptors in man was shown to increase as well vasopressin (AVP), which acts synergistically with CRH as pituitary-adrenocortical axis stimulator during stress. Copeptin (CoP), the C-terminal part of pre-pro-AVP, is released in an equimolar ratio to AVP. It is more stable in the circulation and easier to determine than AVP and it was found to closely mirror the production of AVP. So far, CoP secretion has not been characterized during panic provocation. In 30 healthy male human subjects, we repeatedly measured CoP in plasma during a panic challenge and studied its correlation to Acute Panic Inventory (API) ratings and plasma ACTH and cortisol. CoP levels correlated positively with the increase in API ratings (r=0.41, p=0.03), while ACTH or cortisol did not (r=0.08, p=0.68 and r=0.12, p=0.53, respectively). CoP levels correlated also positively with ACTH (r=0.48, p=0.009) and cortisol (r=0.48, p=0.01) concentrations throughout the CCK-4 challenge. As expected, we found a positive correlation between plasma ACTH and cortisol levels (r=0.57, p=0.001). A vasopressinergic activation during CCK-4 induced panic was demonstrated, which was correlated positively to panic symptoms and pituitary-adrenocortical release. Our findings suggest a role of CoP as a potential surrogate marker of CCK-4 panic symptoms. Further studies are needed to replicate our results and to further clarify the role of CoP as a stress-sensitive hormone in different panic paradigms as well as in panic patients.

Metabolic Actions of the Type 1 Cholecystokinin Receptor: Its Potential as a Therapeutic Target.

Cholecystokinin (CCK) regulates appetite and reduces food intake by activating the type 1 CCK receptor (CCK1R). Attempts to develop CCK1R agonists for obesity have yielded active agents that have not reached clinical practice. Here we discuss why, along with new strategies to target CCK1R more effectively. We examine signaling events and the possibility of developing agents that exhibit ligand-directed bias, to dissociate satiety activity from undesirable side effects. Potential allosteric sites of modulation are also discussed, along with desired properties of a positive allosteric modulator (PAM) without intrinsic agonist action as another strategy to treat obesity. These new types of CCK1R-active drugs could be useful as standalone agents or as part of a rational drug combination for management of obesity.

Elimination of a cholecystokinin receptor agonist 'trigger' in an effort to develop positive allosteric modulators without intrinsic agonist activity.

Cholecystokinin (CCK) acts at the type 1 cholecystokinin receptor (CCK1R) to elicit satiety and is a well-established drug target for obesity. To date, small molecule agonists have been developed, but have failed to demonstrate adequate efficacy in clinical trials, and concerns about side effects and potential toxicity have limited further development of full agonists. The use of positive allosteric modulators (PAMs) without intrinsic agonist activity that are active only for a brief period of time after a meal might represent a safer alternative. Here, we propose a possible novel strategy to develop such compounds by modifying the agonist 'trigger' of an existing small molecule agonist. We have studied analogues of the 1,5-benzodiazepine agonist, GI181771X, in which the N1-isopropyl agonist 'trigger' was modified. While agonist activity was greatly reduced in these compounds, they acted as negative, rather than positive modulators. The parent drug was also found to exhibit no positive modulation of CCK action. Receptor structure-activity relationship studies demonstrated that the mode of docking these derivatives was distinct from that of the parent compound, perhaps explaining their action as negative allosteric modulators. We conclude that this outcome is likely characteristic of the parental agonist, and that this strategy may be more successfully utilized with a parental ago-PAM, possessing intrinsic positive modulatory activity.

Simultaneous quantification of the glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) receptor agonists in rodent plasma by on-line solid phase extraction and LC-MS/MS.

Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) and the cholecystokinin-1 receptor (CCK1-R) have therapeutic potential because of their marked anorexigenic and weight lowering effects. Furthermore, recent studies in rodents have shown that co-administration of these agents may prove more effective than treatment either of the peptide classes alone. To correlate the pharmacodynamic effects to the pharmacokinetics of these peptide drugs in vivo, a sensitive and robust bioanalytical method is essential. Furthermore, the simultaneous determination of both analytes in plasma samples by a single method offers obvious advantages. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is well suited to this goal through its ability to simultaneously monitor multiple analytes through selected reaction monitoring (SRM). However, it is a challenge to find appropriate conditions that allow two peptides with widely disparate physiochemical properties to be simultaneously analyzed while maintaining the necessary sensitivity for their accurate plasma concentrations. Herein, we report an on-line solid phase extraction (SPE) LC-MS/MS method for simultaneous quantification of the CCK1-R agonist AC170222 and the GLP-1R agonist AC3174 in rodent plasma. The assay has a linear range from 0.0975 to 100ng/mL, with lower limits of quantification of 0.0975ng/mL and 0.195ng/mL for AC3174 and AC170222, respectively. The intra- and inter-day precisions were below 15%. The developed LC-MS/MS method was used to simultaneously quantify AC3174 and AC170222, the results showed that the terminal plasma concentrations of AC3174 or AC170222 were comparable between groups of animals that were administered with the peptides alone (247±15pg/mL of AC3174 and 1306±48pg/mL of AC170222), or in combination (222±32pg/mL and 1136±47pg/mL of AC3174 and AC170222, respectively). These data provide information on the drug exposure to aid in assessing the combination effects of AC3174 and AC170222 on rodent metabolism.

Species- and dose-specific pancreatic responses and progression in single- and repeat-dose studies with GI181771X: a novel cholecystokinin 1 receptor agonist in mice, rats, and monkeys.

Compound-induced pancreatic injury is a serious liability in preclinical toxicity studies. However, its relevance to humans should be cautiously evaluated because of interspecies variations. To highlight such variations, we evaluated the species- and dose-specific pancreatic responses and progression caused by GI181771X, a novel cholecystokinin 1 receptor agonist investigated by GlaxoSmithKline for the treatment of obesity. Acute (up to 2,000 mg/kg GI181771X, as single dose) and repeat-dose studies in mice and/or rats (0.25-250 mg/kg/day for 7 days to 26 weeks) showed wide-ranging morphological changes in the pancreas that were dose and duration dependent, including necrotizing pancreatitis, acinar cell hypertrophy/atrophy, zymogen degranulation, focal acinar cell hyperplasia, and interstitial inflammation. In contrast to rodents, pancreatic changes were not observed in cynomolgus monkeys given GI181771X (1-500 mg/kg/day with higher systemic exposure than rats) for up to 52 weeks. Similarly, no GI181771X treatment-associated abnormalities in pancreatic structure were noted in a 24-week clinical trial with obese patients (body mass index >30 or >27 kg/m(2)) as assessed by abdominal ultrasound or by magnetic resonance imaging. Mechanisms for interspecies variations in the pancreatic response to CCK among rodents, monkeys, and humans and their relevance to human risk are discussed.

Characteristics of the cholecystokinin-induced depolarization of pacemaking activity in cultured interstitial cells of Cajal from murine small intestine.

BACKGROUND/AIMS: In this study, we studied the effects of cholecystokinin (CCK) on pacemaker potentials in cultured interstitial cells of Cajal (ICCs) from mouse small intestine using the whole cell patch clamp technique. METHODS: ICCs are pacemaker cells that exhibit periodic spontaneous depolarization, which is responsible for the production of slow waves in gastrointestinal smooth muscle, and generate periodic pacemaker potentials in current-clamp mode. RESULTS: Exposure to CCK (100 nM-5 µM) decreased the amplitudes of pacemaker potentials and depolarized resting membrane potentials. To identify the type of CCK receptors involved in ICCs, we examined the effects of CCK agonists and found that the addition of CCK1 agonist (A-71323, 1 µM) depolarized resting membrane potentials, whereas exposure to CCK2 agonist (gastrin, 1 µM) had no effect on pacemaker potentials. To confirm these results, we examined the effects of CCK antagonists and found that pretreatment with CCK1 antagonist (SR 27897, 1 µM) blocked CCK-induced effects. However, pretreatment with CCK2 antagonist (LY 225910, 1 µM) did not. Furthermore, intracellular GDPßS suppressed CCK-induced effects. To investigate the involvements of phospholipase C (PLC), protein kinase C (PKC), and protein kinase A (PKA) in the effects of CCK in cultured ICCs, we used U-73122 (an active PLC inhibitor), chelerythrine (a PKC inhibitor), SQ-22536 (an inhibitor of adenylate cyclase), or mPKAI (an inhibitor of myristoylated PKA). All inhibitors blocked the CCK-mediated effects on pacemaker potentials. In addition, we found that transient receptor potential classical 5 (TRPC5) channel was involved in CCK-activated currents in cultured ICCs. CONCLUSION: These results suggest that the CCK induced depolarization of pacemaking activity occurs in a G-protein-, PLC-, PKC-, and PKA-dependent manner via CCK1 receptor and TRPC5 channel is a candidate for CCK-activated currents in cultured ICCs in murine small intestine. Therefore, the ICCs are targets for CCK and their interaction can affect intestinal motility.

Calcium handling in porcine coronary endothelial cells by gastrin-17.

In porcine coronary artery endothelial cells (PCAEC), gastrin-17 has recently been found to increase nitric oxide (NO) production by the endothelial NO synthase (eNOS) isoform through cholecystokinin 1/2 (CCK1/2) receptors and the involvement of protein kinase A (PKA), PKC and the ß2-adrenoreceptor-related pathway. As eNOS is the Ca(2)(+)-dependent isoform of the enzyme, we aimed to examine the effects of gastrin-17 on Ca(2)(+) movements. Thus, experiments were performed in Fura-2-acetoxymethyl-ester-loaded PCAEC, where changes of cytosolic Ca(2)(+) ([Ca(2)(+)]c) caused by gastrin-17 were analysed and compared with those of CCK receptors and ß2-adrenoreceptors agonists/antagonists. In addition, some experiments were performed by stimulating cells with gastrin-17 in the presence or absence of cAMP/PKA activator/inhibitor and of phospholipase C (PLC) and Ca(2)(+)-calmodulin dependent protein kinase II (CaMKII) blockers. The results have shown that gastrin-17 can promote a transient increase in [Ca(2)(+)]c mainly originating from an intracellular pool sensitive to thapsigargin and from the extracellular space. In addition, the response of cells to gastrin-17 was increased by the adenylyl cyclase activator and the ß2-adrenoreceptor agonists and affected mainly by the CCK2 receptor agonists/antagonists. Moreover, the effects of gastrin-17 were prevented by ß2-adrenoreceptors and CaMKII blockers and the adenylyl cyclase/PKA and PLC inhibitors. Finally, in PCAEC cultured in Na(+)-free medium or loaded with the plasma membrane Ca(2)(+) pump inhibitor, the gastrin-17-evoked Ca(2)(+) transient was long lasting. In conclusion, this study shows that gastrin-17 affected intracellular Ca(2)(+) homeostasis in PCAEC by both promoting a discharge of an intracellular pool and by interfering with the operation of store-dependent channels through mainly CCK2 receptors and PKA/PLC- and CaMKII-related signalling downstream of ß2-adrenoreceptor stimulation.

Chemical cholecystokinin receptor activation protects against obesity-diabetes in high fat fed mice and has sustainable beneficial effects in genetic ob/ob mice.

The current study has determined the ability of (pGlu-Gln)-CCK-8 to counter the development of diet-induced obesity-diabetes and examined persistence of beneficial metabolic effects in high fat and ob/ob mice, respectively. Twice daily injection of (pGlu-Gln)-CCK-8 in normal mice transferred to a high fat diet reduced energy intake (p < 0.001), body weight (p < 0.01), circulating insulin and LDL-cholesterol (p < 0.001) and improved insulin sensitivity (p < 0.001) as well as oral and intraperitoneal (p < 0.001) glucose tolerance. Energy intake, body weight, circulating insulin and glucose tolerance of (pGlu-Gln)-CCK-8 mice were similar to lean controls. In addition, (pGlu-Gln)-CCK-8 prevented the effect of high fat feeding on triacylglycerol accumulation in liver and muscle. Interestingly, (pGlu-Gln)-CCK-8 significantly (p < 0.001) elevated pancreatic glucagon content. Histological examination of the pancreata of (pGlu-Gln)-CCK-8 mice revealed no changes in islet number or size, but there was increased turnover of beta-cells with significantly (p < 0.001) increased numbers of peripherally located alpha-cells, co-expressing both glucagon and GLP-1. Beneficial metabolic effects were observed similarly in ob/ob mice treated twice daily with (pGlu-Gln)-CCK-8 for 18 days, including significantly reduced energy intake (p < 0.05), body weight (p < 0.05 to p < 0.01), circulating glucose (p < 0.05 to p < 0.01) and insulin (p < 0.05 to p < 0.001) and improved glucose tolerance (p < 0.05) and insulin sensitivity (p < 0.001). Notably, these beneficial effects were still evident 18 days following cessation of treatment. These studies emphasize the potential of (pGlu-Gln)-CCK-8 for the treatment of obesity-diabetes.