Gene expression profiling of gastric mucosa in mice lacking CCK and gastrin receptors.

The stomach produces acid, which may play an important role in the regulation of bone homeostasis. The aim of this study was to reveal signaling pathways in the gastric mucosa that involve the acid secretion and possibly the bone metabolism in CCK1 and/or CCK2 receptor knockout (KO) mice. Gastric acid secretion was impaired and the ECL cell signaling pathway was inhibited in CCK2 receptor KO mice but not in CCK1 receptor KO mice. However, in CCK1+2 receptor double KO mice the acid secretion in response to pylorus ligation-induced vagal stimulation and the ECL cell pathway were partially normalized, which was associated with an up-regulated pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1). The basal part of the gastric mucosa expressed parathyroid hormone-like hormone (PTHLH) in a subpopulation of likely ECL cells (and possibly other cells) and vitamin D3 1α hydroxylase probably in trefoil peptide2-immunoreactive cells. In conclusion, mice lacking CCK receptors exhibited a functional shift from the gastrin-CCK pathways to the neuronal pathway in control of the ECL cells and eventually the acid secretion. Taking the present data together with previous findings, we suggest a possible link between gastric PTHLH and vitamin D and bone metabolism.

CCK1-receptor stimulation protects against gut mediator-induced lung damage during endotoxemia.

BACKGROUND/AIMS: Cholecystokinin 1-receptor (CCK1-R) activation by long chain fatty acid (LCFA) absorption stimulates vago-vagal reflex pathways in the brain stem. The present study determines whether this reflex also activates the cholinergic anti-inflammatory pathway, a pathway known to modulate cytokine release during endotoxemia. METHODS: Mesenteric lymph was obtained from wild type (WT) and CCK1-R knockout (CCK1-R(-/-)) mice intraperitoneally challenged with Lipopolysaccharid (LPS) (endotoxemic lymph, EL) and intestinally infused with vehicle or LCFA-enriched solution. The lymph was analyzed for TNFα, IL-6 and IL-10 concentration and administered to healthy recipient mice via jugular infusion. Alveolar wall thickness, myeloperoxidase (MPO) and TUNEL positive cells were determined in lung tissue of recipient mice. RESULTS: LCFA infusion in WT mice reduced TNFα concentration in EL by 49% compared to vehicle infusion, but had no effect in CCK1-R(-/-) mice. EL significantly increased the alveolar wall thickness, the number of MPO-positive and TUNEL-positive cells compared to control lymph administration. LCFA infusion in WT, but not in CCK1R(-/-) mice, significantly reduced these pathological effects of EL. CONCLUSION: During endotoxemia enteral LCFA absorption reduces TNFα release into mesenteric lymph and attenuates histomorphologic parameters of lung dysfunction. Failure to elicit this effect in CCK1R(-/-) mice demonstrates that anti-inflammatory properties of LCFAs are mediated through CCK1-Rs.

Alterations in activity and energy expenditure contribute to lean phenotype in Fischer 344 rats lacking the cholecystokinin-1 receptor gene.

CCK is hypothesized to inhibit meal size by acting at CCK1 receptors (CCK1R) on vagal afferent neurons that innervate the gastrointestinal tract and project to the hindbrain. Earlier studies have shown that obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which carry a spontaneous null mutation of the CCK1R, are hyperphagic and obese. Recent findings show that rats with CCK1R-null gene on a Fischer 344 background (Cck1r(-/-)) are lean and normophagic. In this study, the metabolic phenotype of this rat strain was further characterized. As expected, the CCK1R antagonist, devazepide, failed to stimulate food intake in the Cck1r(-/-) rats. Both Cck1r(+/+) and Cck1r(-/-) rats became diet-induced obese (DIO) when maintained on a high-fat diet relative to chow-fed controls. Cck1r(-/-) rats consumed larger meals than controls during the dark cycle and smaller meals during the light cycle. These effects were accompanied by increased food intake, total spontaneous activity, and energy expenditure during the dark cycle and an apparent reduction in respiratory quotient during the light cycle. To assess whether enhanced responsiveness to anorexigenic factors may contribute to the lean phenotype, we examined the effects of melanotan II (MTII) on food intake and body weight. We found an enhanced effect of MTII in Cck1r(-/-) rats to suppress food intake and body weight following both central and peripheral administration. These results suggest that the lean phenotype is potentially driven by increases in total spontaneous activity and energy expenditure.

Development of obesity in the Otsuka Long-Evans Tokushima Fatty rat.

Understanding the early factors affecting obesity development in males and females may help to prevent obesity and may lead to the discovery of more effective treatments for those already obese. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat model of obesity is characterized by hyperphagia-induced obesity, due to a spontaneous lack of CCK(1) receptors. In the present study, we focused on the behavioral and physiological aspects of obesity development from weaning to adulthood. We examined body weight, feeding efficiency, fat pad [brown, retroperitoneal, inguinal and epydidimal (in males)] weight, inguinal adipocyte size and number, leptin and oxytocin levels, body mass index, waist circumference, and females' estrous cycle structure. In the males, central hypothalamic gene expression was also examined. OLETF rats presented overall higher fat and leptin levels, larger adipocytes, and increased waist circumference and BMI from weaning until adulthood, compared with controls. Analysis of developmental patterns of gene expression for hypothalamic neuropeptides revealed peptide-specific patterns that may underlie or be a consequence of the obesity development. Analysis of the developmental trajectories toward obesity within the OLETF strain revealed that OLETF females developed obesity in a more gradual manner than the males, presenting delayed obesity-related "turning points," with reduced adipocyte size but larger postweaning fat pads and increased adipocyte hyperplasia compared with the males. Intake decrease in estrus vs. proestrus was significantly less in OLETF vs. Long-Evans Tokushima Otsuka females. The findings highlight the importance of using different sex-appropriate approaches to increase the efficacy of therapeutic interventions in the treatment and prevention of chronic early-onset obesity.

Normal feeding and body weight in Fischer 344 rats lacking the cholecystokinin-1 receptor gene.

A large body of evidence has demonstrated that one mechanism by which cholecystokinin (CCK) inhibits food intake through activation of CCK1 receptors (CCK1R) on vagal afferent neurons that innervate the gastrointestinal tract and project to the hindbrain. OLETF rats, which carry a spontaneous null mutation of the CCK1R, are hyperphagic, obese, and predisposed to type 2 diabetes. Recently, by introgressing the OLETF-derived, CCK1R-null gene onto a Fischer 344 genetic background, we have been able to generate a CCK1R-deficient, congenic rat strain, F344.Cck1r(-/-), that in contrast to OLETF rats, possesses a lean and normoglycemic phenotype. In the present study, the behavioral and neurobiological phenotype of this rat strain was characterized more fully. As expected, intraperitoneal injections of CCK-8 inhibited intake of chow and Ensure Plus and induced Fos responses in the area postrema and the gelatinosus, commissural and medial subdivisions of the nucleus tractus solitarius of wild-type F344.Cck1r(+/+) rats, whereas CCK-8 was without effect on food intake or Fos induction in the F344.Cck1r(-/-) rats. F344.Cck1r(-/-) and F344.Cck1r(+/+) rats did not differ in body weight and showed comparable weight gain when maintained on Ensure Plus for 2 weeks. Also, no difference was found in 24-h food intake, and dark-phase meal frequency or meal size between F344.Cck1r(+/+) and F344.Cck1r(-/-) rats. As expected, blockade of endogenous CCK action at CCK1R increased food intake and blocked the effects of peripheral CCK-8 in wild-type F344.Cck1r(+/+) rats. These results confirm that in rats with a F344 background, CCK-1R mediates CCK-8-induced inhibition of food intake and Fos activation in the hindbrain and demonstrate that selective genetic ablation of CCK1R is not associated with altered meal patterns, hyperphagia, or excessive weight gain on a palatable diet.

Cholecystokinin regulates expression of Y2 receptors in vagal afferent neurons serving the stomach.

The intestinal hormones CCK and PYY3-36 inhibit gastric emptying and food intake via vagal afferent neurons. Here we report that CCK regulates the expression of Y2R, at which PYY3-36 acts. In nodose ganglia from rats fasted up to 48 h, there was a fivefold decrease of Y2R mRNA compared with rats fed ad libitum; Y2R mRNA in fasted rats was increased by administration of CCK, and by refeeding through a mechanism sensitive to the CCK1R antagonist lorglumide. Antibodies to Y2R revealed expression in both neurons and satellite cells; most of the former (89 +/- 4%) also expressed CCK1R. With fasting there was loss of Y2R immunoreactivity in CCK1R-expressing neurons many of which projected to the stomach, but not in satellite cells or neurons projecting to the ileum or proximal colon. Expression of a Y2R promoter-luciferase reporter (Y2R-luc) in cultured vagal afferent neurons was increased in response to CCK by 12.3 +/- 0.1-fold and by phorbol ester (16.2 +/- 0.4-fold); the response to both was abolished by the protein kinase C inhibitor Ro-32,0432. PYY3-36 stimulated CREB phosphorylation in rat nodose neurons after priming with CCK; in wild-type mice PYY3-36 increased Fos labeling in brainstem neurons but in mice null for CCK1R this response was abolished. Thus Y2R is expressed by functionally distinct subsets of nodose ganglion neurons projecting to the stomach and ileum/colon; in the former expression is dependent on stimulation by CCK, and there is evidence that PYY3-36 effects on vagal afferent neurons are CCK dependent.

Reduced CCK-induced Fos expression in the hindbrain, nodose ganglia, and enteric neurons of rats lacking CCK-1 receptors.

Many of the actions of cholecystokinin (CCK) are mediated by CCK-1 receptors, expressed by enteric and vagal afferent neurons. Otsuka Long-Evans Tokushima Fatty rats (OLETF) do not express CCK-1 receptors, and do not exhibit the vagally mediated responses to CCK. To determine whether the OLETF rat's failure to respond to CCK is correlated with failure of CCK to activate enteric and vagal neurons, we quantified neuronal Fos immunoreactivity in the dorsal vagal complex of the hindbrain, the nodose ganglia, and the ganglia of the myenteric and submucosal plexuses of the duodenum following intraperitoneal injection of CCK-8 (20 microg/kg). Compared to vehicle injection, CCK administration resulted in significant increases in the number of Fos-immunopositive neurons in the nucleus of the solitary tract, area postrema, and dorsal vagal motor nucleus of control, LETO rats. In OLETF rats, however, CCK did not increase numbers of Fos-immunoreactive neurons in any of these brain structures. CCK also induced significantly larger numbers of Fos-immunoreactive neuronal nuclei in the nodose ganglia of LETO rats, but not in the nodose ganglia of OLETF rats. Finally, LETO, but not OLETF rats exhibited striking increases in the number of Fos-immunoreactive nuclei of myenteric and submucosal neurons, following CCK injection. Absence of CCK-induced Fos expression in OLETF rats is consistent with attenuation of ingestive and gastrointestinal responses to CCK in the CCK-1 receptor deficient rats. These results also suggest that CCK-induced Fos expression in enteric and vagal sensory neurons of rats can be accounted for entirely by activation of CCK-1 receptors and is not due to occupation of CCK-2 (gastrin) receptors, which also are expressed in the intestine and by some vagal afferent neurons.

Effects of cholecystokinin (CCK)-8 on hypothalamic oxytocin-secreting neurons in rats lacking CCK-A receptor.

Peripheral administration of cholecystokinin (CCK)-8 selectively activates oxytocin (OXT)-secreting neurons in the supraoptic (SON) and the paraventricular nuclei (PVN) with the elevation of plasma OXT level in rats. We examined the effects of intravenous (iv) administration of CCK-8 on the neuronal activity of hypothalamic OXT-secreting neurons and plasma OXT level in Otsuka Long-Evans Tokushima Fatty (OLETF) rats that have a congenital defect in the expression of the CCK-A receptor gene. In situ hybridization histochemistry (ISH) for c-fos mRNA revealed that the expression of the c-fos gene was not induced in the SON, the PVN, the nucleus of the tractus solitarius (NTS) and the area postrema (AP) 30 min after iv administration of CCK-8 (20 and 40 microg/kg) in OLETF rats. In Long-Evans Tokushima Otsuka (LETO) rats (controls), c-fos mRNA was detected abundantly in those nuclei 30 min after iv administration of CCK-8 (20 microg/kg). Immunohistochemistry for c-fos protein (Fos) showed that the distributions of Fos-like immunoreactivity (LI) were identical to the results obtained from ISH. Dual immunostaining for OXT and Fos revealed that Fos-LI was mainly observed in OXT-secreting neurons in the SON and the PVN of LETO rats 90 min after iv administration of CCK-8 (20 microg/kg). Radioimmunoassay for OXT and arginine vasopressin (AVP) showed that iv administration of CCK-8 did not cause significant change in the plasma OXT and AVP levels in OLETF rats, while iv administration of CCK-8 caused a significant elevation of plasma OXT level without changing the plasma AVP level in LETO rats. These results suggest that peripheral administration of CCK-8 may selectively activate the hypothalamic OXT-secreting neurons and brainstem neurons through CCK-A receptor in rats.

Preventive and therapeutic effects of the protease inhibitor camostat on pancreatic fibrosis and atrophy in CCK-1 receptor-deficient rats.

OBJECTIVES: Recent studies have demonstrated that synthetic protease inhibitors could ameliorate the progression of pancreatic fibrosis in some animal models. Since oral administration of protease inhibitors increases the plasma cholecystokinin (CCK) levels and causes hypertrophy of the pancreas in rats, there is a possibility that the protease inhibitor inhibits fibrosis in the pancreas via endogenous CCK release. We examined the effects of camostat, a synthetic protease inhibitor, on histopathologic changes in Otsuka Long-Evans Tokushima Fatty (OLETF) rat that has genetically no expression of CCK-1 receptor and displays inflammation and degeneration of the pancreas. METHODS: Three groups of OLETF rats received a camostat-rich diet (200 mg/100 g normal diet) from 12 to 28 weeks of age or from 12 or 28 weeks of age to the age of 72 weeks, while the fourth group received standard rat diet. RESULTS: Pancreatic wet weight and pancreatic contents of protein, DNA, amylase, lipase, and trypsin in camostat-treated rats were significantly higher than those in the untreated control rats. Immunohistochemical studies of the pancreas showed that expressions of interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and alpha-smooth muscle actin in camostat-treated rats were greatly suppressed compared with those in the untreated control rats. Atrophy and fibrosis in the pancreas observed in the untreated control rats were not found in camostat-fed rats. CONCLUSION: The results of the present study suggest that camostat greatly inhibits pancreatic inflammation and prevents and reverses fibrosis and atrophy of the pancreas in the genetically obese and CCK-1 receptor-deficient OLETF rats.

Treatment for hyperglycemia promotes pancreatic regeneration in rats without CCK-1 receptor gene expression.

INTRODUCTION AND AIM: Recent studies have suggested that CCK is not essential for normal pancreatic growth in mice. We examined whether the treatment of hyperglycemia participates in a non-CCK-1-receptor-mediated mechanism of pancreatic regeneration after partial (30%) pancreatectomy (Px) with use of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model for type 2 diabetes mellitus without CCK-1 receptor gene expression. METHODOLOGY: Male OLETF rats were divided into five groups at 24 weeks of age. The first group was killed to examine the pancreas at 24 weeks of age (PrePx). The second group underwent a midline laparotomy and received a standard rat chow (ShamPx). The remaining three groups of rats received one of the following three treatments after Px: a standard rat chow (PxC), a diet containing acarbose (PxA), or a standard rat chow and once-daily subcutaneous injection of insulin (PxI) for 8 weeks. RESULTS: PxC rats had significantly higher serum glucose levels than did PxA and PxI rats. Pancreatic weight and pancreatic contents of protein in PxA and PxI rats were significantly higher than in PxC rats. The pancreas in PxC rats was atrophic, and marked inflammatory cell infiltration was observed in the pancreas. In addition, tumor necrosis factor-alpha (TNFalpha) was expressed in the inflammatory cells, acinar cells, and islets in PxC rats. However, histologic alterations, including expression of TNFalpha, remained at a minimum in PxA and PxI rats. CONCLUSION: We conclude that the control of serum glucose levels plays an important role in preventing pancreatic atrophy and participates in the non-CCK-1-receptor-mediated mechanisms of pancreatic growth in rats.