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Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.

We previously reported that ingestion of 60 mL of red wine or vodka prior to the ingestion of a pancake significantly inhibited the gastric emptying of the pancake in male subjects, but not in female subjects, and that the retention times of wine and vodka were significantly longer than those of the congener of red wine and mineral water in male subjects, whereas in female subjects the retention times of these four drinks did not differ significantly from one another. We hypothesized that the menstrual cycle may influence the gastric emptying of alcohol beverages. Here, we determined and compared the retention times of vodka and water in the stomach during the luteal phase and the follicular phase. Ten female healthy volunteers were studied. They recorded their basal body temperatures every day, and participated in the following experiments: each volunteer drank mineral water or vodka containing 14% alcohol (60 mL) during the low-temperature (follicular) phase as well as during the high-temperature (luteal) phase. The retention time of vodka was significantly longer than that of mineral water during the follicular phase, but no significant differences between the retention times of the two drinks were observed during the luteal phase. In conclusion, the menstrual cycle influences the gastric emptying rate of alcohol.

Several studies on alcohol and gastric emptying using the (13)C breath test showed that alcohol consumption delayed gastric emptying of meals in healthy male subjects. However, they did not employ female subjects, and the retention time of alcoholic beverages in the stomach has not been examined, yet. We examined the retention time (= gastric emptying rate) of alcoholic beverages in the stomach in healthy male and female subjects. We also examined whether the congeners (nonalcoholic components) of red wine have any effect on gastric emptying. The retention time of 60 mL of red wine, vodka, congeners of red wine, or mineral water, was measured using a (13)C labeled acetic acid breath test. In male subjects, the retention time of wine and vodka was significantly longer than that of congeners and mineral water. In female subjects, although the (13)C content in the breath was slightly but significantly decreased by wine and congeners, but not by vodka, and the parameters for gastric emptying did not differ significantly among the 4 drinks. That is, alcohol hardly influenced the retention time in female subjects. In conclusion, there are sex differences in the gastric emptying rate of alcohol.

Aging is associated with a progressive decrease in appetite and food intake. We focused on the age-associated changes of the stimulatory effect of the appetite-regulating peptide, ghrelin. Food intake and the concentrations of acyl ghrelin and desacyl ghrelin in the plasma and in the stomach were measured with and without overnight fasting in young and old mice. Moreover, the food intake in response to the intraperitoneal administration of graded doses of acyl ghrelin was compared between young and old mice. Fasting drives food intake in young mice, but not in old mice. The concentrations of acyl ghrelin and desacyl ghrelin in the plasma and in the stomach were higher in the old mice than in the young mice. Food intake did not increase in old mice when stimulated by the administration of 1-3 nmol of acyl ghrelin, which could produce a significant increase in food intake in young mice. In conclusion, food intake did not increase in old mice after either overnight fasting or the administration of acyl ghrelin. The release and synthesis of ghrelin seem to be rather higher in old mice compared to young mice. These increases might be the results of compensation for the decline of receptor (and/or post-receptor) functions., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Inflammatory process is involved in the pathogenesis of diabetic nephropathy. In this article, we show that cholecystokinin (CCK) is expressed in the kidney and exerts renoprotective effects through its anti-inflammatory actions. DNA microarray showed that CCK was upregulated in the kidney of diabetic wild-type (WT) mice but not in diabetic intracellular adhesion molecule-1 knockout mice. We induced diabetes in CCK-1 receptor (CCK-1R) and CCK-2R double-knockout (CCK-1R(-/-),-2R(-/-)) mice, and furthermore, we performed a bone marrow transplantation study using CCK-1R(-/-) mice to determine the role of CCK-1R on macrophages in the diabetic kidney. Diabetic CCK-1R(-/-),-2R(-/-) mice revealed enhanced albuminuria and inflammation in the kidney compared with diabetic WT mice. In addition, diabetic WT mice with CCK-1R(-/-) bone marrow-derived cells developed more albuminuria than diabetic CCK-1R(-/-) mice with WT bone marrow-derived cells. Administration of sulfated cholecystokinin octapeptide (CCK-8S) ameliorated albuminuria, podocyte loss, expression of proinflammatory genes, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, CCK-8S inhibited both expression of tumor necrosis factor-α and chemotaxis in cultured THP-1 cells. These results suggest that CCK suppresses the activation of macrophage and expression of proinflammatory genes in diabetic kidney. Our findings may provide a novel strategy of therapy for the early stage of diabetic nephropathy.

Gallstone disease is one of the most prevalent digestive diseases. The frequency of gallstone disease is about 10% in middle-age persons and 20% in aged persons. Gallbladder dysmotility and stasis of bile flow promote sludge and/or gallstone formation. Gallbladder contraction depends on cholecystokinin (CCK) via CCK-1 receptors (R)s. Previously, we raised CCK-1R deficient (-/-) mice and observed sludge and/or gallstone formation in more than 30% at 12-24 months of age. As ursodeoxycholate (UDCA) is commonly used for patients with gallstone disease, we expected that continuous administration of UDCA could prevent sludge and/or gallstone formation in CCK- 1R(-/-) mice. A diet containing 0.1% UDCA was administered in age-matched CCK-1R(-/-) and wild-type male and female mice for 8 months. Administration of UDCA decreased the frequency of sludge and/or gallstone formation compared with the control (CRF-1) diet (39%→26% in male, 35%→25% in female mice); however, these effects did not attain a level of statistical significance. Although the body weight was significantly higher in UDCA-fed than CRF-1-fed male mice regardless of genotypes, the plasma lipid concentrations did not differ between the two diets. In conclusion, administration of UDCA was less effective than expected at preventing sludge and/or gallstone formation in CCK-1R(-/-) mice.

Objectives: Pancreatic functions were determined in a Ki-ras-induced actin-interacting protein (KRAP)-deficient (-/-) mouse mutant., Methods: Pancreatic enzyme, protein, and DNA contents were measured, and histological examinations were conducted. The mixture of bile-pancreatic juice was collected, and amylase and bile acid outputs were determined. Oral glucose tolerance test was determined. Moreover, the gene expression of KRAP was determined in cholecystokinin (CCK)-A(1) receptor (-/-) mice., Results: The body weight was smaller, and the ratio of pancreatic wet weight/body weight was higher in KRAP(-/-) mice compared with wild-type mice. The enzyme contents, but not DNA content, in the pancreas of KRAP(-/-) mice were higher than those of wild-type mice. Histological examination revealed the increase in the number of zymogen granules in the pancreatic acinar cells of KRAP(-/-) mice. Amylase secretions in response to CCK-octapeptide sulfate were significantly higher in KRAP(-/-) than wild-type mice, whereas the basal secretion did not differ between the 2 genotypes. A normal glucose tolerance was observed in KRAP(-/-) mice. The gene expression of KRAP in CCK-A(1) receptor (-/-) mice was significantly lower than in wild-type mice., Conclusions: The lack and/or decrease in KRAP level in the pancreas may promote the pancreatic growth and hypertrophy.

Aims: Most of the acetaldehyde, a recognized animal carcinogen, generated during alcohol metabolism is eliminated by liver mitochondrial aldehyde dehydrogenase 2 (ALDH2). More than 40% of Japanese people have the inactive form of ALDH2, and inactive ALDH2 is a risk factor for multiple cancer of the esophagus, as well as head and neck cancer. Possible associations between pancreatic cancer and ALDH2 gene polymorphism, as well as between colon cancer and ALDH2 gene polymorphism, in conjunction with smoking and/or drinking habits, were examined in a Japanese population., Methods: Patients with pancreatic cancer (n = 187) and with colon cancer (n = 49) were examined. The drinking (5 g ethanol consumption/day) and/or smoking habits as well as ALDH2 gene polymorphism were examined. The age-matched control subjects were recruited in the NILS Longitudinal Study of Aging (LSA)., Results: Aging, smoking and inactive ALDH2, but not alcohol, are independent risk factors for pancreatic cancer. The frequency of smoking habits tended to be higher in patients with colon cancer compared with the patients without cancer. However, age, body mass index or the distribution of ALDH2 genotypes did not differ significantly among the patients with colon cancer, colon polyps and others., Conclusions: Inactive ALDH2 is an independent risk factor for pancreatic cancer, but inactive ALDH2 might not be a risk for colon cancer.

Aging is associated with a progressive decrease in appetite and food intake. The reasons for the decline in food intake are multifactorial, and relate to both peripheral and central mechanisms. Current studies about the regulation of food intake suggest that there are many central mediators that control the appetite. To determine the mechanism of age-associated decrease in appetite and food intake, we focused on the age-associated changes of the suppressing and stimulatory effect of some appetite-regulating peptides. At first, we examined cholecystokinin (CCK), one of the typical appetite-suppressing factors. Although sensitivity to CCK is enhanced in old animals, the mechanism underlying this effect has not been elucidated. Next, we focused on the appetite-stimulating peptides, orexin-A, neuropeptide Y (NPY) and ghrelin, which are known to play a critical role in food intake. To determine the age-associated decrease in appetite and food intake, we compared the stimulatory effect of intracerebroventricular administration of orexin-A, NPY and ghrelin. We report the studies of the age-associated changes of appetite-regulating peptides in this review.

Background: Alcoholic beverages stimulate gastric acid secretion and increase the appetite. Although ingested ethanol stimulates pancreatic secretion, alcoholic beverages contain several congeners. N-methyltyramine (NMT) was isolated from beer as a factor in stimulating gastric acid secretion. In this study, we examined NMT to determine whether the congener stimulated pancreatic secretion in conscious rats., Methods: Cannulae were inserted into male Wistar rats to separately drain bile and pancreatic secretions: 2 duodenal cannulae, a gastric cannula, and an external jugular vein cannula. The rats were placed in modified Bollman-type restraint cages. After a 4-day recovery period, experiments were conducted on unanesthetized rats. Different concentrations of NMT (5, 25, and 50 microg/kg) solutions were infused into the stomach. To examine the mechanism, the effects of the proton pump inhibitor, cholecystokinin (CCK-BR) antagonist (YM022), CCK-AR antagonist (CR1505), and atropine were administered prior to the NMT (25 microg/kg) infusion. The effect of intravenous infusion of NMT (7.5 microg/kg) was then determined. Moreover, dispersed acini were prepared, and the effect of different concentrations of NMT on amylase release was determined., Results: Intragastric administration of NMT significantly increased pancreatic exocrine secretion in a dose-dependent manner. Atropine eliminated the stimulatory effect of NMT, but the infusion of the proton pump inhibitor, YM022, and CR1505 did not. Intravenous infusion of NMT did not affect pancreatic secretion, and NMT did not stimulate amylase release in vitro., Conclusions: N-methyltyramine stimulates pancreatic secretion via the cholinergic gastro-pancreatic reflex. The NMT content in beer was 2 mg/l, so that if a person weighing 60 kg consumes a 750 ml of beer, 25 microg/kg NMT will be ingested. Therefore, the stimulatory effect of beer on pancreatic secretion was produced not only by ethanol but also by the congener, NMT.

Objective: The aim of this study was to evaluate the efficacy and toxicities of the gemcitabine and paclitaxel combination regimen as second-line chemotherapy for patients with advanced or metastatic urothelial carcinoma (UC) who have previously been treated with platinum-based chemotherapy for the metastatic disease., Methods: Thirty-three patients with advanced or metastatic UC who had received platinum-based chemotherapy were treated with an outpatient gemcitabine and paclitaxel combination regimen. A dose of 180 mg/m(2) paclitaxel was administered by intravenous (IV) infusion on Day 1, and 1000 mg/m(2) gemcitabine was administered by IV on Days 1, 8 and 15.The course was repeated every 28 days. Patients were evaluated after every 2 cycles of therapy using computed tomography., Results: Of the 33 patients enrolled in this study, 30 could be evaluated to determine treatment efficacy; 10 had an objective response [overall response rate: 33.3%, 95% confidence interval (CI), 19.2-51.2%]. The median overall survival was 11.3 months (95% CI, 7.2-13.6 months). The chemotherapy sensitivity differed with disease site. The response rates of lung and bone metastases were 27% and 14%, and the progressive disease (PD) rates of lung and bone metastases were 13% and 14%, respectively. On the other hand, the response rate of liver metastasis was 14%, and its PD rate was 57%. None of the patients (n = 3) with adrenal metastasis responded to this regimen. Toxicities were mild, and no life-threatening complications occurred., Conclusions: Gemcitabine and paclitaxel combination therapy is a tolerable and active regimen for patients with advanced UC after failure of platinum-based chemotherapy.

Obesity and related metabolic disorders have become leading causes of adult morbidity and mortality. KRAP (Ki-ras-induced actin-interacting protein) is a cytoskeleton-associated protein and a ubiquitous protein among tissues, originally identified as a cancer-related molecule, however, its physiological roles remain unknown. Here we demonstrate that KRAP-deficient (KRAP(-/-)) mice show enhanced metabolic rate, decreased adiposity, improved glucose tolerance, hypoinsulinemia and hypoleptinemia. KRAP(-/-) mice are also protected against high-fat diet-induced obesity and insulin resistance despite of hyperphagia. Notably, glucose uptake in the brown adipose tissue (BAT) in KRAP(-/-) mice is enhanced in an insulin-independent manner, suggesting that BAT is involved in altered energy homeostasis in KRAP(-/-) mice, although UCP (Uncoupling protein) expressions are not altered. Of interest is the down-regulation of fatty acid metabolism-related molecules, including acetyl-CoA carboxylase (ACC)-1, ACC-2 and fatty acid synthase in the liver of KRAP(-/-) mice, which could in part account for the metabolic phenotype in KRAP(-/-) mice. Thus, KRAP is a novel regulator in whole-body energy homeostasis and may be a therapeutic target in obesity and related diseases.

Gastrin is important for stimulating acid secretion as well as differentiating gastric mucosal cells via cholecystokinin-2 receptors (CCK-2Rs). In turn, CCK acts preferably via CCK-1R to release somatostatin, and somatostatin has been postulated to exhibit a tonic inhibition of gastrin bioactivity. The present study was designed to examine the hypothesis that CCK-1R and 2R may act in opposite directions in gastric acid secretion. Having generated CCK-1R(-/-), 2R(-/-), and 1R(-/-)2R(-/-) mice, we examined the regulation of gastric acid secretion in four genotypes including wild-type mice. Parietal cells possess histamine receptors, muscarinic receptors, and CCK-2Rs. Since histamine increases cAMP and carbachol increases calcium, the responses of gastric acid secretion to graded doses of histamine, carbachol, and a combination of histamine + carbachol were determined. The sensitivity to histamine did not differ among the four genotypes, while the maximal acid secretion was lower in CCK-2R(-/-) mice than in wild-type mice. In addition, sensitivity to carbachol was impaired in mice without CCK-2R. The interaction of histamine and carbachol was conserved in all genotypes. In conclusion, CCK-2R is necessary to respond to carbachol as well as to produce the maximal acid secretion, while the role of CCK-1R in acid secretion is less important.

Daily behaviors are strongly dominated by internally generated circadian rhythms, but the underlying mechanisms remain unclear. In mammals, photoentrainment of behaviors to light-dark cycles involves signaling from both intrinsically photosensitive retinal ganglion cells and classic photoreceptor pathways to the suprachiasmatic nucleus (SCN). How classic photoreceptor pathways work with the photosensitive ganglion cells, however, is not fully understood. Although cholecystokinin (CCK) peptide has been shown to be present in a variety of vertebrate retinas, its function at a systems level is also unknown. In the present study we examined a possible role of CCK-A receptors in photoentrainment using CCK-A receptor knockout mice. The lacZ reporter gene within a gene-knockout cassette revealed precise localization of CCK-A receptors in the circadian clock system. We demonstrated that CCK-A receptors were located predominately on glycinergic amacrine cells but were rarely found on SCN neurons. Moreover, Ca(2+) imaging analysis demonstrated that the CCK-A agonist, CCK-8 sulfate (CCK-8s), mobilized intracellular Ca(2+) in amacrine cells but not glutamate-receptive SCN neurons. Furthermore, light pulse-induced mPer1/mPer2 gene expression in SCN, behavioral phase shifts, and the pupillary reflex were significantly reduced in CCK-A receptor knockout mice. These data indicate a novel function of CCK-A receptors in the nonimage-forming photoreception presumably via amacrine cell-mediated signal transduction pathways.

Gastric acid secretion in response to chemical stimulation and to mechanical stimulation was investigated in adult and old mice. The protein expression of a proton pump (H(+)-K(+)-ATPase), a marker of parietal cell function, was determined by Western blotting. Acid secretion was stimulated by histamine (500 and 1000 microg/kg) or carbachol (10 and 20 microg/kg). To investigate the response to mechanical stimulation, the stomach was distended by an intragastric injection of isotonic saline (0.5, 1.0, 1.5, and 2.0 ml). Administration of two doses of histamine produced a dose-dependent increase in acid secretion in adult mice, whereas a higher dose of histamine failed to produce a further increase in old mice. Gastric acid secretion, whether produced by carbachol or mechanical stimulation, did not differ between the two age groups. The protein expression of H(+)-K(+)-ATPase was significantly lower in old mice than in adult. Insofar as histamine increases acid secretion via the cyclic AMP (cAMP) pathway in parietal cells, while carbachol and gastric distention do so via the calcium signaling pathway, the cAMP signaling pathway may be more susceptible to aging than the calcium signaling pathway. The decrease in the secretory capacity of acid secretion in the old mice may be partly attributable to a decrease in parietal cell function, as shown by decrease in H(+)-K(+)-ATPase protein expression.

Background: The frequency of Japanese subjects over 20 years old with metabolic syndrome is 45.6% in men but just 16.7% in women. The reason why Japanese male subjects are more susceptible to metabolic syndrome than women is unknown. One possibility is the higher frequency of Japanese male subjects (40-70 years old) who had a drinking habit (67%), while that of female subjects was only 25%. In addition, daily fat intake was markedly increased in Japanese subjects (from 9% to 25%), and cholesterol cholelithiasis is one of the most rapidly increasing digestive diseases during the past 50 years. The object of this study is to examine whether a potential sex-related risk factor exists in the manifestation of metabolic syndrome as well as gallstone formation., Methods: Gallbladder dysmotility accerelates gallstone formation and gallbladder contraction depends on cholecystokinin (CCK) and its receptor (CCK-1R). We developed CCK-1R gene knockout (-/-) mice. The effects of the fat- and protein- enriched diet OA-2 on body weight, hyperlipidemia, and frequencies of sludge and gallstone formation were examined, and compared between wild-type and CCK-1R(-/-) male and female mice. The OA-2 diet contains slightly higher protein and fat (7.9 % fat and 27.6 % protein) compared with a standard diet (CRF-1) (5.6 % fat and 22.6 % protein), but their total energies are similar. After weaning, CRF-1 was provided until 3 months of age in all animals. Administration of an OA-2 diet was started when age-matched CCK-1R(-/-) and wild-type male and female mice reached maturity, at 3 months of age. Administration of CRF-1 was continued in the rest of the animals. Mice were sacrificed by guillotine at 6 and 12 months of age and the blood was collected to measure plasma levels of triglyceride and cholesterol. The gallbladder was removed and classified as normal (clear gallbladder), clouded (sludge formation), and/or containing gallstone formations., Results: As long as CRF-1 was provided, the frequency of sludge and/or gallstone formation in CCK-1R(-/-) male mice was 3 of 8 (35%) and 4 of 9 (45%) in females at 12 months of age, whereas no gallstone formation was observed at 6 months of age. On the other hand, male mice fed OA-2 increased their body weight and plasma lipid concentrations, compared with those fed CRF-1 regardless of genotype. Under the OA-2 diet, sludge and gallstone formation was observed at 6 months of age, not only in CCK-1R(-/-) male mice but also in wild-type male mice. In contrast, parameters in female mice did not differ between the two diets., Conclusion: Male mice were more susceptible to protein- and fat-enriched diet-induced obesity than female mice, and hyper-nutritional status accelerated sludge and gallstone formation in male mice.

Background: Gallbladder dysmotility accelerates cholelithiasis. In turn, gallbladder dysmotility can occur secondary to inflammation and excess cholesterol accumulation in gallbladder smooth muscle., Methods: The present study was designed to determine how much gallbladder dysmotility contributes to gallstone formation as a primary cause and whether a sex difference exists in gallstone formation by comparing cholecystokinin-1 receptor gene-deficient [CCK-1R(-/-)] male and female mice., Results: No sludge or gallstone formation was observed in mice at 6 months of age. The frequency of sludge and gallstone formation in mice at 12 and 24 months of age was slightly higher in female CCK-1R(-/-) mice than in males, but the difference was not significant., Conclusions: Gallbladder dysmotility may have accelerated sludge and gallstone formation, but its contribution was limited. A 12-month period was required to produce gallstones, and after the mice reached 12 months of age, further ageing did not increase the frequency of gallstones. The effect of sex did not reach a significant level.

Since the isolation and sequencing of cholecystokinin (CCK), considerable advances have been made in understanding the roles played by this peptide as a hormone and as a neuropeptide. CCK-1(A) and 2(B) receptor (R) cDNAs have been cloned; shortly thereafter, the naturally occurring CCK-1R gene-deficient rat (the Otsuka Long-Evans Tokushima Fatty (OLETF) rat) was discovered. This strain develops adult-onset diabetes with obesity, and has a 6847 base-pair deletion of the CCK-1R gene in which the promoter lesion and the first two exons are missing. At the same time, the genomic structures of CCK-1R in rats, mice, and humans were clarified. The CCK-1R gene consists of five exons interrupted by four introns. It has been determined that there is species- and tissue-specific CCK receptor heterogeneity of expression; in particular, there is evidence that the human pancreas does not express CCK-1R, while the pancreas in rodents primarily expresses CCK-1R. Although CCK-1R polymorphisms with amino acid changes such as 21Gly to Arg, 71 Arg to Gly, and 364 Val to Ile were discovered in subjects with obesity and diabetes mellitus, these changes occur sporadically. We identified two sequence changes, a G to T change in nucleotide -128, and an A to G change in nucleotide -81, in the promoter region of the CCK-1R gene. This polymorphism is considered to be a single nucleotide polymorphism (SNP) related to weight control difficulties in obese subjects as well as to psychiatric disorders. The precise molecular mechanisms of this polymorphism remain to be clarified.

Cholecystokinin receptors (CCK-Rs) have been classified into two subtypes: CCK-AR (1R) and -BR (2R). We generated CCK-AR(-/-), CCK-BR(-/-), and CCK-AR(-/-)BR(-/-) mice and found that the gastric emptying of a liquid meal was increased in CCK-BR(-/-) and AR(-/-)BR(-/-) mice, compared with wild-type and CCK-AR(-/-) mice. Given that enhanced gastric emptying leads to eating, food intake after overnight fasting was examined, as was the effect of CCK-8S on food intake. Male mice 6-8 months of age were deprived of food for 16 h with free access to water, after which they were injected intraperitoneally (0.1 ml/mouse) with either vehicle or CCK-8 (0.3, 1.0, or 3.0 nmol/mouse), and their food intake was monitored for 4 h. CCK-8S inhibited food intake in wild-type and CCK-BR(-/-) mice, but not in CCK-AR(-/-) or AR(-/-)BR(-/-) mice. Unexpectedly, we observed a lower food intake in CCK-AR(-/-)BR (-/-) mice treated with vehicle than in mice of the other genotypes. To examine the mechanism of decrease in food intake in CCK-AR(-/-)BR(-/-) mice, the involvement of ghrelin was determined in wild-type and CCK-AR(-/-)BR(-/-) mice. Fasting plasma ghrelin levels were significantly lower in CCK-AR (-/-)BR(-/-) mice than in wild-type mice, and no increase in response to fasting was observed in CCK-AR(-/-)BR(-/-) mice. An administration of acyl-ghrelin produced a small increase in food intake in CCK-AR(-/-)BR(-/-) mice, but not to the levels of wild-type mice. In conclusion, CCK-AR(-/-)BR(-/-) mice showed lower food intake as well as lower response to exogenous ghrelin, and a lower plasma ghrelin level after fasting, though which receptor is more important is unknown.

To clarify the pharmacological mechanism of ezetimibe, SCH 58053, an analog of ezetimibe, was intraduodenally administered to lymph-fistula rats, and its effect on lymphatic lipid transport in the intestine was monitored. SCH 58053, 5.0 mg/kg body weight, was administered one hour before a 5-hour infusion of a lipid emulsion containing 40 micromol/h of triolein and 2.74 micromol/h of cholesterol (Experiment 1) or co-administered at 5.0 mg/kg body weight/h with the lipid emulsion for 4 hours to rats that had been infused with the lipid emulsion alone for 3 hours (Experiment 2). SCH 58053 administration significantly inhibited lymphatic cholesterol transport, but not triglyceride transport, in both groups compared to control rats that did not receive SCH 58053. The ratio of free cholesterol to total cholesterol in the lymph of the treated rats was unchanged compared to the control rats. Thus, the results showed that SCH 58053 is a potent, rapid, and selective inhibitor of lymphatic cholesterol transport in the intestine.

Otsuka Long-Evans Tokushima Fatty (OLETF) rats lack cholecystokinin-A receptor (CCK-AR) because of a genetic abnormality. We observed that body temperature homeostasis in response to changes in ambient temperature was deteriorated in OLETF rats, while the functions of the signal outputs from the hypothalamus to effectors were not impaired. Deteriorated homeostasis was also seen in CCK-AR deficient (-/-) mice. In the present study, we examined whether the sensory pathway involved in transmitting signals about temperature from the skin to the brain was impaired in OLETF rats. To elucidate the involvement of CCK-AR function, we conducted the same experiment in CCK-AR(-/-) mice. Responses to thermal pain were assessed using the Hargreaves' plantar test apparatus. Shortening of withdrawal latency was observed in OLETF rats compared to control rats, indicating thermal hyperalgesia. Behavioral responses following paw withdrawal were disturbed in OLETF rats. The 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid contents in the hippocampus and frontal cortex of OLETF rats were significantly higher than in those of the controls. CCK-AR(-/-) mice did not show any differences from wild-type mice. In conclusion, OLETF rats showed thermal hyperalgesia and disturbed responses to thermal pain, and an alteration of 5-HT function might have a role in this disturbance.

Objectives: SH-2 containing inositol 5'-phosphatase 2 (SHIP2) is a family of inositol 5'-phosphatases, which possess the 5'-phosphatase activity that hydrolyzes phosphatidylinositol-3, 4, 5-trisphosphate to phosphatidylinositol-3, 4-bisphosphate and is suspected to negatively regulates the metabolic signaling of insulin. To clarify the possible involvement of SHIP2 in physiological abnormalities, we examined the human SHIP2 gene polymorphism in a Japanese cohort., Methods: We searched single-nucleotide polymorphisms (SNPs) on the human SHIP2 gene promoter and 5'-untranslated region (5'-UTR) and investigated their relationship with impaired fasting glycemia (IFG) in a Japanese cohort. Next, the effect of the SNPs on promoter activity was examined in HeLa and HL60 cells., Results: Among the several SNPs detected on the human SHIP2 gene promoter and 5'-UTR, 3 SNPs (-405 C/A, +57 G/A, and +334 C/T) formed the haplotypes CGC and AAT and were found at a relatively high frequency in the Japanese population. The frequency of genotypes (+334 CT and TT) was significantly higher in the group with IFG than in the normal group (P < 0.0001, odds ratio = 2.23, 95% confidence interval = 1.50-3.32). This association was not affected by age and gender. Furthermore, one haplotype (+57 A, +334 T) which was inserted into a luciferase reporter plasmid and existed more frequently in the IFG group than in the normal group exhibited increased promoter activity in the culture cells compared with the other haplotype (+57 G, +334 C)., Conclusions: The SNPs in the SHIP2 gene promoter and the 5'-UTR may account partly for the IFG and may be a marker for the risk of diabetes.

(-)Deprenyl (D) has been shown to be effective in prolonging life span in experimental animals, although, there are some discrepancies in its effect on the life span the even within the same species (rats). The present study aims to clarify the reason for these discrepancies. Male F344/DuCrj rats began receiving subcutaneous (s.c.) injections of D at the age of 18 months. Doses used were 0.25, 0.50, and 1.0 mg/kg/injection (inj.), three times a week. Average life spans of animals were significantly longer in male rats given 0.25 and 0.5 mg/kg/inj.; however, rats given a 1.0 mg/kg dose began dying earlier than control rats, leading to an inverse U-shaped dose-efficacy relationship, a hormesis. Old (27-month-old) rats given different doses of D for 1 month showed a typical hormetic response for antioxidant enzyme activities, indicating a significant increase in superoxide dismutase (SOD) and catalase (CAT) activities in brain dopaminergic regions with four lower doses (0.25 to 2 mg/kg/inj., 3 times a week), but a significantly negative response with the highest dose (4 mg/kg/inj.). Our results clearly indicate that a proper dose of D within a certain dose range can significantly increase the life span of rats, but that a greater dose becomes less effective and may actually adversely affect the life span of rats. A similar hormetic response for its effect on antioxidant enzyme activities and the parallel between the two different effects of D suggest a possible causal relationship between these two effects of D. The presence of this effective dose range of D may explain previously reported discrepancies in the effect of D on the life span of animals.

In mammals, including humans, a brain-gut hormone, cholecystokinin (CCK) mediates the satiety effect via CCK-A receptor (R). We generated CCK-AR gene-deficient (-/-) mice and found that the daily food intake, energy expenditure, and gastric emptying of a liquid meal did not change compared with those of wild-type mice. Because CCK-AR(-/-) mice show anxiolytic status, we examined the effects of restraint stress. Seven hours of restraint stress was found to significantly decrease both body weight and food intake during the subsequent 3 days in all tested animals. On the fourth day after restraint stress, the CCK-AR(-/-) mice showed a significantly higher level of daily food intake than prior to stress, and food intake recovered to prestress levels in the wild-type mice. Since peripheral CCK-AR has been known to mediate gastric emptying, both gastric emptying and gastric acid secretion were determined to examine the mechanism of overeating in CCK-AR(-/-) mice. Neither gastric emptying nor gastric acid secretion differed between CCK-AR(-/-) and wild-type mice on the fourth day after stress. In contrast, however, the contents of dopamine and its metabolites in the cerebral cortex of CCK-AR(-/-) mice were increased by stress, but were rather decreased in wild-type mice. Changes in 5-hydroxytryptamine (5-HT) and its metabolite 5HIAA did not differ between the genotypes. In conclusion, CCK-AR(-/-) mice showed overeating after restraint stress, and dopaminergic hyperfunction in the brain of these mice was observed. The present evidence suggests that the CCK-AR function, possibly via altering the dopaminergic function, might be involved in overeating after stress.

Aims: Cholecystokinin (CCK) modulates dopamine release in the nucleus accumbens through the CCK-A receptor (CCK-AR). The dopaminergic neurotransmission between the ventral tegmental area and the limbic forebrain is a critical neurobiological component of alcohol and drug self-administration. Based on the evidence of interaction between CCK and dopamine, we had found previously that the CCK-AR gene -81A/G polymorphism was associated with alcohol dependence. Since the precise mechanism underlying this association has not been elucidated, the role of CCK-AR in ethanol ingestion was examined using CCK-AR gene deficient (-/-) mice and compared with those of CCK-BR(-/-) and wild-type mice., Methods: The two-bottle choice protocol was conducted and the righting reflex was examined in these three genotypes. Furthermore, the protein level of dopamine 2 receptor (D2R) in the nucleus accumbens was determined by western blotting., Results: CCK-AR(-/-) mice consumed more ethanol than CCK-BR(-/-) and wild-type mice, and showed no aversion to high concentrations of ethanol solution. However, the difference was actually in the total fluid consumption and alcohol preference remained unchanged, indicating that the differences were not specific to alcohol. Behavioral sensitivity to ethanol, examined using the righting reflex, did not differ significantly between the groups. D2R expression in the nucleus accumbens was significantly lower in the CCK-BR(-/-) mice and was significantly higher in CCK-AR(-/-) mice than in wild-type mice., Conclusions: Voluntary ingestion of ethanol differed between CCK-AR(-/-) and CCK-BR(-/-) mice. The difference might be attributable in part to the different levels of D2R expression in the nucleus accumbens.

Objectives: Alcohol abuse causes pancreatic damage in humans. However, only 5% of alcoholic patients have a clinical manifestation of pancreatitis, and the genetic predisposition of alcohol-associated pancreatitis remains elusive. Nonoxidative metabolites of ethanol, fatty acid ethyl esters (FAEEs), might play an important role in pancreatic damage. Carboxylester lipase (CEL) has been known to catalyze FAEE synthesis from fatty acids and ethanol., Methods: The variable number of tandem repeat (VNTR) polymorphism in the coding region of the CEL gene was studied in patients with alcoholic pancreatitis (n = 100), in alcoholics without pancreatitis (n = 52), in patients with nonalcoholic pancreatitis (n = 50), in hyperlipidemia patients (n = 96), and control subjects (n = 435)., Results: The frequency of the NN-type (wild-type) gene was significantly decreased in patients with alcoholic pancreatitis than in other groups. The frequency of subjects who had the L allele in patients with alcoholic pancreatitis was significantly higher than in other groups. The distribution of the CEL gene polymorphism was not different among the control subjects, alcoholics without pancreatitis, patients with nonalcoholic pancreatitis, and patients with hyperlipidemia., Conclusions: The CEL gene polymorphism, especially an increase in the frequency of the L allele, was found to be associated with alcohol-induced pancreatitis.

We have characterized the regulation of human SH-2 containing inositol 5'-phosphatase 2 (SHIP2) gene expression. First, the transcription initiation sites and the sequence of the 5' upstream region of human SHIP2 gene were elucidated. Next, the minimal promoter of the human SHIP2 gene was identified by reporter gene assays in HL60 cells and differentiated human subcutaneous white adipocytes. An Sp1 element proximal to the transcription initiation site was indispensable for full promoter activity and bound specifically by Sp1 and Sp3 proteins. These findings suggest that human SHIP2 gene expression, like other housekeeping genes, is controlled by the Sp-family of transcription factors.

Objectives: Most of the acetaldehyde, a recognized animal carcinogen, generated during alcohol metabolism is eliminated by liver mitochondrial aldehyde dehydrogenase 2 (ALDH2). More than 40% of Japanese have the inactive form of ALDH2, and inactive ALDH2 is a risk factor for multiple cancers of the esophagus as well as head and neck cancer. Possible associations between pancreatic cancer and ALDH2 gene polymorphism, in conjunction with smoking and/or drinking habits, were examined in a Japanese population., Methods: We investigated 114 patients (70 male and 44 female) with pancreatic cancer and compared them with 2070 control subjects (1050 male and 1020 female). The drinking (5 g ethanol consumption/d) and/or smoking habits as well as ALDH2 gene polymorphism were examined., Results: In male subjects, the frequency of the active form of ALDH2 (2*1/2*1) was lower in pancreatic cancer patients than in control subjects (P = 0.018). The frequency of subjects with both smoking and drinking habits was significantly higher in pancreatic cancer patients than in control subjects having ALDH2*1/2*1 and ALDH2*1/2*2. The frequency of smoking habit alone was significantly higher in pancreatic cancer patients compared with control subjects having inactive ALDH2. Drinking habit had no relation to pancreatic cancer. In female subjects, neither habit had a relation to pancreatic cancer., Conclusions: Smoking habit did increase the risk of pancreatic cancer, and this risk was further enhanced in subjects with inactive ALDH2 in a male population but not in a female population. There was no relationship between drinking habit and pancreatic cancer in either sex population.

Purpose: To study the association between Cholecystokinin A receptor (CCKAR) genotypes and intelligence in community-living men and women., Method: Subjects were 2251 community-dwelling Japanese men and women aged 40 to 79 years. The CCKAR gene promoter polymorphisms A-81G and G-128T were determined. Intelligence was assessed by Japanese Wechsler Adult Intelligence Scales - Revised Short Forms (JWAIS-R SF). The difference in intelligence between wild type and mutation was tested., Results: There were no subjects with AA/GT, AA/TT, or AG/TT genotypic combinations. Both A-81G and G-128T genotypes were related to intelligence quotient (IQ) estimated by JWAIS-R SF. The mean and SE of IQ levels of subjects with the wild-type allele and the mutation allele at nucleotide -128 were 103.4 +/- 0.3 and 101.6 +/- 0.6, respectively. There was a significant difference in IQ for G-128T (p=0.008). The difference in IQ for A-81G was also significant (p=0.011). The IQ level was 103.6 +/- 0.4 in the subjects with the wild-type allele and 102.0 +/- 0.5 in the subjects with the mutation. Differences in IQ levels by haplotypes for combinations of A-81G/G-128T were examined. IQ significantly decreased with an increasing number of mutation alleles (p=0.018)., Conclusion: There were statistically significant differences in IQ for CCKAR gene promoter polymorphisms A-81G and G-128T in community-living Japanese.

Objectives: Although many studies have investigated the acute effect of ethanol on the rat pancreas, the effect of intravenous injection of ethanol on pancreatic secretion has been controversial. The purposes of this study were to examine whether intraduodenal and intravenous administration of ethanol stimulates pancreatic secretion in Wistar rats. To elucidate the mechanism, we examined whether ethanol influences pancreatic secretion in CCK-A receptor-deficient rats., Methods: Rats were prepared with cannulae for draining bile and pancreatic juice separately. The experiments were conducted in unanesthetized rats. Different concentrations of ethanol solutions were infused at the rate of 1.5 mL/h into the duodenum, and half concentrations of the respective ethanol solutions were prepared and infused into the duodenum at a rate of 3 mL/h. Then we determined the pancreatic responses to simultaneous administration of a liquid meal with ethanol. Moreover, the effect of intravenous infusion of ethanol on pancreatic secretion was examined. Finally, the effect of intraduodenal and intravenous infusion of ethanol was determined in CCK-A receptor-deficient rats., Results: Intraduodenal infusion of ethanol significantly increased pancreatic secretion, and these responses depended on the amount of ethanol instilled into the lumen, except for 3% ethanol solution, which did not affect pancreatic secretion. Simultaneous administration of a liquid meal with ethanol augmented pancreatic response in rats treated with the higher 2 ethanol concentrations (33% and 66%). Intravenous administration of ethanol slightly but significantly increased pancreatic secretion. However, no stimulation was observed in CCK-A receptor-deficient rats regardless of ethanol administration route., Conclusions: Both intraduodenal and intravenous administration of ethanol stimulated pancreatic secretion, and this stimulation required CCK-A receptor.

Aging is associated with a progressive decrease in appetite and food intake. The appetite-stimulating peptides orexin A, neuropeptide Y (NPY) and ghrelin are known to play a critical role in food intake. In this study, the stimulatory effect of intracerebroventricular administration of these peptides on food intake was compared among young (4 months old), adult (11 months old) and old (24-27 months old) male Wistar rats. A stainless steel cannula was implanted stereotactically into the left lateral ventricle. After a 7-day recovery period, different doses of orexin A (0.25-3 nmol), NPY and ghrelin (0.03-1 nmol) were injected into the left lateral ventricle without anesthesia. Food consumption was measured at 1, 2 and 4 h after injection. We also examined the plasma levels of acylated and desacyl ghrelin in young and old rats by ELISA. Intracerebroventricular administration of orexin A and NPY stimulated food intake in young and adult rats, but no effects were observed at any dose in old rats. Ghrelin increased food intake in a dose-dependent manner in all groups, and the effect of ghrelin was reduced with advancing age. Neither the acylated nor the desacyl plasma ghrelin level differed significantly between young and old rats. In conclusion, the orexigenic effect of the peptides orexin A, NPY and ghrelin decreased in old rats, and this reduction may have been responsible for the age-related decrease in food intake.

Aging is associated with a progressive decrease in appetite and food intake. Both A and B orexins, expressed in specific neurons of the lateral hypothalamic area, have been implicated in the regulation of sleep and feeding. In this study, the stimulatory effect of intracerebroventricular administration of the orexins on food intake was compared between young (4-mo-old) and old (25- to 27-mo-old) male Wistar rats. A stainless steel cannula was implanted stereotactically into the left lateral ventricle. After a 7-day recovery period, different doses (0-30 nmol) of orexins were injected into the left lateral ventricle without anesthesia. Food and water consumptions were measured at 1, 2, and 4 h after injection. The protein levels of orexin receptors, a specific receptor for orexin-A (OX1R) and a receptor for both orexin-A and -B (OX2R), in the hypothalamus were determined by Western blot analysis and compared between young and old rats. Intracerebroventricular administration of orexin-A stimulated food intake in a dose-dependent manner in young rats. However, no effects were observed at any dose in old rats. The protein level of OX1R in the hypothalamus was significantly lower in old rats than in young rats, although the protein level of OX2R was comparable between groups. Results of the present study indicate that the function of the orexin system is diminished in old rats. The decrease in the OX1R protein level in the hypothalamus could be responsible for orexin-A's lack of stimulation of food intake in old rats.

The circadian rhythm of the body core temperature (T(c)) and the effects of changes in ambient temperatures on the homeostasis of T(c) in Otsuka Long Evans Tokushima Fatty (OLETF) rats, which are naturally occurring cholecystokinin (CCK)-A receptor (CCK-AR) gene knockout (-/-) rats, were examined. In addition, the peripheral responses to warming or cooling of the preoptic and anterior hypothalamic region (PO/AH) were determined. The circadian rhythm of T(c) in OLETF rats was similar to that in Long-Evans Tokushima (LETO) rats; this rhythm was characterized by a higher T(c) during the dark period and a lower T(c) during the light period. When the ambient temperature was changed within the limits of 0 degrees C to 30 degrees C, the changes in T(c) of LETO rats were associated with the changes in ambient temperature, whereas those in OLETF rats were dissociated from the temperature changes. The OLETF rats showed a large hysteresis. The peripheral responses to warming or cooling of PO/AH, including shivering of the neck muscle and changes in skin temperature of the tail and footpad, were similar in OLETF and LETO rats. To confirm the role of CCK-AR in the regulation of body temperature, the values of T(c) in the CCK-AR(-/-) mice were compared with those in CCK-B receptor (CCK-BR) (-/-), CCK-AR(-/-)BR(-/-), and wild-type mice. In the mice, the circadian rhythms of T(c) were the same, regardless of the genotype. Mice without CCK-AR showed larger hysteresis than mice with CCK-AR. From these results, we conclude that the lack of CCK-AR causes homeostasis of T(c) in rats and mice to deteriorate.

We investigated the relationship of polymorphisms in the cholecystokinin 1 receptor [CCK1R; G to T (n-128), A to G (n-81)] and the beta3-adrenergic receptor (beta3-AR; Trp64Arg) with midlife weight gain. The participants were 1012 Japanese men and women (40 to 59 years of age). Their weight at 18 years old was obtained from a questionnaire. Weight change was defined as the current weight minus the weight at 18 years old. Subjects were grouped into four categories by these genotypes: W/W=noncarriers, W/H=Arg64 carriers of the beta3-AR, H/W=T (n-128) or G (n-81) carriers of the CCK1R, H/H=T (n-128) or G (n-81) and Arg64 carriers. In men, the interaction between the CCK1R and beta3-AR polymorphisms was significant (two-way ANOVA, p < 0.05), but neither the CCK1R nor the beta3-AR was individually associated with weight gain. The H/H group showed a higher possibility of weight gain of 10 kg or more compared with the W/W group in men. The odds ratio for weight gain (> or =10 kg) of H/H was 2.54 (95% confidence interval: 1.50 to 4.30) compared with W/W. In women, neither main effect nor interaction was significant. These results suggest that the combination of CCK1R and the beta3-AR polymorphisms is a contributing factor for midlife weight gain in men.

Although the idea that energy metabolism of rats decreases with age has been widely accepted, few studies with regard to the diurnal rhythm of energy expenditure have been reported. Whether age alone altered the diurnal rhythm of energy expenditure was examined in Sprague-Dawley (SD) rats. The same determination was conducted in Otsuka Long Evans Tokushima Fatty (OLETF) rats to examine the effect of insulin resistance and diabetes. OLETF rats were developed as a model of non-insulin-dependent diabetes mellitus (NIDDM) with mild obesity. The characteristic features of OLETF rats are late onset of hyperglycemia at about 18 weeks of age, followed by insulin deficiency at about 65 weeks. Age-associated changes in diurnal rhythm of energy expenditure were not observed in SD rats. In OLETF rats, the diurnal rhythm of energy expenditure with two peaks was observed at 8 weeks of age, while these two peaks disappeared at 24 weeks of age (with NIDDM). Then, the pattern of diurnal rhythm at 44 weeks of age (with advanced NIDDM) was resembled to that of 62 weeks of age (with insulin deficiency). In conclusion, we clarified the changes in diurnal rhythm of energy expenditure associated with the progress of diabetes, while age alone did not alter the diurnal rhythm., (Copyright 2004 Elsevier Ireland Ltd.)

Several lines of evidence have suggested that naturally occurring alterations in cholecystokinin (CCK) systems could contribute to the development of panic disorder (PD). Among recent investigations, polymorphisms of the CCK and CCK-B receptor (R) genes were investigated, but the results were inconclusive. We recently cloned the genomic structures of human CCK-AR, and determined the transcriptional start site of the human CCK-AR gene. Two sequence changes were detected in the promoter region: a G to T change in nucleotide -128 and an A to G change in nucleotide -81 (GenBank database under accession number D85606). The frequencies of the genotypes and haplotypes of these two polymorphisms were compared in 109 Japanese patients with PD and 400 age- and gender-matched normal Japanese control subjects. The frequency of variant genotypes (-81A/G, -128G/T; G/G, G/T, and G/G, T/T) having variant haplotype (-81G/-128T) was significantly higher in PD than in controls (P < 0.0001, OR = 2.81, 95% CI = 1.74-4.39). The statistical differences between the haplotype distributions in the PD and control groups were highly significant: the frequency of variant haplotype (-81G/-128T) was higher in the former group than in the latter (P < 0.0001). This association was not affected by clinical characteristics such as age, gender, and age at onset of PD. In this study, the first to report the positive association of the CCK-AR polymorphisms and PD, haplotype analyses further strengthened the association based on our comparison of genotype distributions. The CCK-AR gene polymorphism may be involved in the neurobiology of PD., (Copyright 2004 Wiley-Liss, Inc.)

Objectives: There are many experimental models for chronic pancreatitis. However, it remains unclear which animal models of pancreatic fibrosis can be categorized as chronic pancreatitis models. We compared the histologic features of some animal models of pancreatic fibrosis/chronic pancreatitis and chronic pancreatitis in humans., Methods and Results: Human chronic pancreatitis due to chronic alcohol abuse and unknown etiology showed interlobular fibrosis and a cirrhosis-like appearance. Histopathologically, spontaneous pancreatitis models, WBN/Kob rats and OLETF rats, showed localized/nodular fibrotic lesions, which consisted of swollen, aggregated, atrophic islets of Langerhans; loss of the exocrine parenchyma and hemosiderin deposition that was seldom distributed in the interlobular area. On the other hand, fibrosis in the canine model, which was produced by combining alcohol administration with incomplete pancreatic duct obstruction, was characterized by interlobular fibrosis admixed with a cirrhosis-like appearance very similar to that in human chronic pancreatitis., Conclusion: Most experimental models for chronic pancreatitis, except alcohol administration combined with other procedures such as incomplete pancreatic duct obstruction, are different from human chronic pancreatitis.

Background: Although cholecystokinin (CCK) has been shown to inhibit gastric emptying via CCK-A receptors (CCK-ARs), the role of CCK-B receptors (CCK-BRs) has not been verified. We examined whether gastric emptying of a nonnutrient liquid load was modified in CCK-AR, BR, and ARBR gene knockout mice., Methods: A liquid gastric load prepared with phenol red was administered via an orogastric tube (0.15 ml/mouse). The animals were killed by decapitation, and gastric emptying was estimated at 10 and 30 min after ingestion. The effects of the sulfated form of CCK-8 (CCK-8S) and of graded doses of atropine were examined. In addition, a proton pump inhibitor was administered to wild-type mice to examine the contribution of gastric acid to emptying., Results: Gastric emptying was significantly enhanced in mice lacking CCK-BR, as compared with wild-type and CCK-AR(-/-) mice. CCK-8S inhibited gastric emptying in mice with CCK-AR, but not in mice without CCK-AR. A proton pump inhibitor did not affect gastric emptying. Atropine dose dependently inhibited gastric emptying in all genotypes. The thickness of smooth muscle was comparable for all genotypes., Conclusions: The gastric emptying of a nonnutrient liquid load was enhanced in mice without CCK-BR, although the precise mechanism is not known. Although cholecystokinin (CCK) has been shown to inhibit gastric emptying via CCK-A receptors (CCK-ARs), the role of CCK-B receptors (CCK-BRs) has not been verified. We examined whether gastric emptying of a nonnutrient liquid load was modified in CCK-AR, BR, and ARBR gene knockout mice.

Aims: Cholecystokinin (CCK), one of the most abundant neurotransmitter peptides, interacts with dopamine. Dopaminergic neurotransmission between the ventral tegmental area and the limbic forebrain is a critical neurobiological component of alcohol and drug self-administration. CCK modulates dopamine release in the nucleus accumbens via the CCK-A receptor (R). We recently determined the transcriptional start site of the human CCK-AR gene, and detected two sequence changes (-81A/G and -128G/T) in the promoter region. The aims of the present study were to determine the prevalence of the -81A/G and -128G/T polymorphism of the CCK-AR gene between alcoholics and normal control subjects and the occurrences of the polymorphisms in subtypes of alcoholics., Methods: The above polymorphisms were examined in 435 alcoholics and 1490 control subjects. We excluded subjects with inactive ALDH2 and employed the subjects with ALDH2*1/2*1 (384 alcoholics and 792 controls)., Results: The allelic frequency of -81G in the CCK-AR gene polymorphism (-81A/G) was significantly higher in alcoholics than in control subjects. However, there were no differences between the two groups with respect to the frequency of -128G/T. Alcoholic patients with antisocial personality disorder and with first-degree alcoholic relatives were significantly associated with a higher frequency of the -81G allele. In addition, the age of onset of alcohol dependence was significantly earlier in patients with this allele., Conclusions: The CCK-AR gene -81A/G polymorphism, especially in the -81G allele, may be associated with intractable alcoholism.

Ghrelin, a novel growth-hormone releasing peptide, was originally isolated from rat and human stomach. Immunohistochemical analyses revealed that ghrelin-immunoreactive neurons were localized in the hypothalamic arcuate nucleus. The function of the digestive organs is controlled from the central nervous system, and the vagus nerve plays an important role. Intracerebroventricular and intravenous administration of ghrelin significantly increased gastric acid secretion, and its effect was abolished by vagotomy. In the present study, the effect of centrally injected ghrelin on pancreatic exocrine secretion was examined in conscious rats. Moreover, an electrophysiologic study was conducted in anesthetized rats to examine whether the excitation of vagal efferent nerve could be induced by ghrelin. To determine the pancreatic exocrine secretion, rats were prepared with cannulae draining bile and pancreatic juice separately. The experiments were conducted in conscious rats on day 4 or 5 after the operation. To exclude the involvement of gastric acid, a proton pump inhibitor omeprazole (5 micromol/kg) was administered into the duodenum 1 h before ghrelin injection. An intracerebroventricular administration of ghrelin (12, 60, and 300 pmol/10 microl) significantly increased pancreatic fluid and protein output in a dose-dependent manner. Pretreatment with the ganglion blocker hexamethonium and with atropine completely abolished the stimulatory effect of central ghrelin. In contrast, an intravenous injection of ghrelin (300 pmol/10 microl) had no effect. Centrally administered ghrelin stimulated the vagal efferent nerve in anesthetized rats. In conclusion, centrally administered ghrelin stimulates pancreatic exocrine secretion through the vagal efferent nerve, and the stimulatory action is independent of gastric acid secretion.

Vinegar is generally believed to be good for health. A mash consisting of 35% ethanolic extract from bitter melon malt vinegar-water (8:50:42) was subjected to further acetate fermentation and the resulting vinegar was converted to dried vinegar powder by spray drying after adsorption on dextrin, which was mixed with a commercial rat chow (CRF-1) in the ratio of 1:19 so as to prepare an experimental diet. Male 12-wk old rats of LETO and OLETF strains were fed this experimental diet in parallel with CRF-1 (control) and examined for respiratory quotient (RQ) and blood or plasma parameters associated with diabetes mellitus. Administration of the experimental diet increased daily food intake as well as daily energy expenditure in both strains. RQ significantly lessened in the vinegar diet-fed group of LETO strain, which was reflected not only in the increased energy consumption from fat but also in the decreased energy consumption from carbohydrate, while no significant difference was observed between both dietary groups of OLETF strain in this respect. The profiles of diurnal energy expenditure in both dietary groups of LETO strain exerted two peaks before lights-on and lights-off. Nevertheless, there was a clear difference between both dietary groups of OLETF strain: interestingly the reproduction of the two peaks became conspicuous in the vinegar diet-fed group despite the lack of such peaks in the control. As a consequence of blood or plasma inspection, it turned out that there was no change in HbA1c but a significant increase in plasma cholesterol in the vinegar diet-fed OLETF rats. From these results, a long-term administration of bitter melon malt vinegar can be expected to suppress a lowering of energy turnover inherent with aging and thereby improve anorexia rather than to bring about a preventive effect against the manifestation of NIDDM.

The etiology of gallstones is multifactorial, with interactions between genes and the environment. We generated cholecystokinin (CCK) -A receptor (R)-deficient (-/-) mice and found that CCK did not produce gallbladder contraction in CCK-AR(-/-) mice. The purpose of this study was to identify the role of CCK-AR on gallstone formation. Age-matched CCK-AR gene (+/+) and (-/-) progenies were used. Sludge and gallstone formation, as well as plasma cholesterol levels, were measured at 12 and 24 months of age. Sludge and gallstone formation were significantly higher in CCK-AR(-/-) mice than in CCK-AR(+/+) mice at 12 and 24 months of age, although these were not different between 12 and 24 months of age. The plasma cholesterol levels, daily food intake, and body weight were not significantly different between CCK-AR(+/+) and (-/-) mice. Sludge and gallstone formation were not observed at 6 months of age. In conclusion, deteriorated gallbladder contraction due to a lack of CCK-AR favored gallstone formation after the middle age of life.

Laboratory rats are normally confined to cages that markedly restrict their physical activity. In these rats, the resting energy expenditure accounts for 90% of the total daily energy expenditure, while the daily physical activity in humans consumes 30% of the total daily energy expenditure. Otsuka Long Evans Tokushima Fatty (OLETF) rats have been developed as a model of non-insulin-dependent diabetes mellitus (NIDDM) with mild obesity, and obesity is an important factor that induces diabetes in this strain. We implemented a running-wheel exercise regimen that was the equivalent of normal physical activity to provide light exercise for OLETF rats. The purpose of the study was to determine if light exercise improves the age-related decline in energy metabolism and glucose intolerance in OLETF rats. The effects were also compared in control Long Evans Tokushima (LETO) rats. From 12 to 46 weeks of age, the rats performed a running-wheel exercise (3000 m/day). Energy metabolism was determined at 8-week intervals. The typical increase in body weight was significantly decreased in OLETF rats in response to exercise, while no significant effect was observed in LETO rats. Energy expenditure and basal metabolic rate (BMR) per kilogram body weight (not whole-body weight) were increased by exercise in OLETF rats, but not in LETO rats. At 46 weeks of age, after exercise, the blood glucose and hemoglobin (Hb)A1c levels, as well as the plasma levels of insulin, triglyceride, cholesterol, and leptin significantly decreased in OLETF rats, while only the plasma levels of cholesterol and leptin significantly decreased in LETO rats. Light exercise thus appears to be beneficial for preventing age-related decline in energy metabolism and glucose intolerance in OLETF rats.

Cholecystokinin (CCK)-A receptor (AR) and B receptor (BR) share highly amino acid sequence homology and overlap in their tissue distribution. We examined the anxiety-related behavior of CCK-AR, CCK-BR, and CCK-ARBR gene knockout (-/-) mice in the elevated plus-maze. CCK-AR(-/-) mice showed a significantly higher frequency of open-arm entries than wild-type and CCK-BR(-/-) mice, whereas the percentage open-arm entry values in CCK-AR(-/-) mice did not differ from those in wild-type mice. Thus, this increased frequency in open-arm entries for CCK-AR(-/-) mice was interpreted to be due to an increase in locomotor activity, rather than to a reduction in anxiety. By contrast, CCK-BR(-/-) mice showed significantly lower percentage open-arm entry values and spent significantly less time in the open- arms than wild-type and CCK-AR(-/-) mice. We therefore conclude that a lack of CCK-BR increases the anxiety-related behavior of the mouse in the elevated plus- maze.

Female C57BL mice of six different ages (from 6 to 26 months) were given a protein free-diet (PFD) for 1 week and then given a normal diet (ND). Mice were examined for enzyme activities of glutathione S-transferase (GST) in the hepatic cytosol fraction using l-chloro-2, 4-dinitrobenzene (CDNB) as substrate. Enzyme activities were very close among the six different age groups when examined for basal levels as well as after 1 week of PFD. However, a remarkable age difference became manifest when animals were examined 2 days after the start of ND refeeding following 1 week of PFD. In young animals (6, 8 months), activities became much higher than their respective basal levels while in old animals (24, 26 months) enzyme levels remained significantly lower than their basal levels on day 2 of ND refeeding. A significant negative linear correlation between enzyme activities (Y axis) and animal age (X axis) was demonstrated only on day 2 or 3 of ND refeeding, while in control animals or animals given 1 week of PFD diet, no significant correlation could be found between enzyme activities and animal age. We conclude that liver cytosolic GST activity is a function of animal age only after a dietary manipulation such as a PFD and ND refeeding, while basal GST activities remain stable throughout the observation period of animal age., (Copyright 2002 Elsevier Science Ireland Ltd.)

Background: The etiology of gallstone formation is multifactorial, and genetic factors are involved. The genetic variations of cholecystokinin A receptor (CCK-AR) in patients having gallstones and the molecular mechanisms of this polymorhpism were examined. The involvement of CCK-AR in gallstone formation was confirmed using CCK-AR gene knockout mice., Methods: CCK-AR gene expression was determined by Northern transfer analysis in gallbladders with or without gallstones. Genetic variations were determined by Southern blot and by direct sequencing. Molecular mechanisms in terms of the transcriptional activity and methylation status were examined. Finally, we investigated whether gallstone formation was enhanced in CCK-AR gene knockout mice., Results: The gene expression of CCK-AR was significantly decreased in gallbladders with gallstones compared to those without gallstones. No genetic variations were detected in the coding region, but two sequence variations were detected in the promoter region in gallstone patients. However, no significant differences were found for the promoter activities of polymorphic promoter constructs. In contrast, less methylation in the promoter region was related to substantial expression of the CCK-AR gene. Gallstone formation was enhanced in CCK-AR gene knockout mice. The homozygote (GG/TT) polymorphism of the CCK-AR gene showed a significantly higher percentage of body fat., Conclusions: Deteriorating gallbladder contractions, possibly induced by alterations in the CCK-AR gene, as well as CCK-AR gene polymorphism, promoted gallstone formation.

Introduction: Digestive organs are controlled from the central nervous system, and the vagus nerve plays an important role. Orexins are recently purified neuropeptides localized in neurons within the lateral hypothalamus., Aim: To examine the effects of centrally injected Orexin-A and B on pancreatic exocrine secretion in conscious rats., Methodology: Rats were prepared with cannulae draining bile and pancreatic juice separately. The experiments were conducted without anesthesia on day 4 or 5 after the operation., Results: Intracerebroventricular administration of Orexin-A (0.25, 0.5, and 1.0 nmol) significantly increased pancreatic fluid and protein output in a dose-dependent manner. A significant stimulatory effect of Orexin-B was not observed. Pretreatment with the ganglion blocker hexamethonium and with atropine completely abolished the stimulatory effect of central Orexin-A. Central Orexin-A significantly increased pancreatic secretion after pretreatment with omeprazole. Intravenous injection of Orexin-A had no effect. Centrally administered Orexin-A stimulated the vagal efferent nerve in anesthetized rats., Conclusions: Centrally administered Orexin-A stimulates pancreatic exocrine secretion through the vagal efferent nerve, and the stimulatory action is independent of gastric acid secretion.

Introduction: In humans, cholecystokinin (CCK) stimulates pancreatic secretion, and CCK-A receptor antagonists prevent it in vivo. However, the human pancreas has been reported to express mainly CCK-B receptors., Aim: To elucidate this discrepancy., Methodology: We prepared dispersed acini from human pancreas and examined whether various doses of CCK stimulated the release of amylase, in comparison with the effects of neuromedin C, carbamylcholine, and secretin., Results: Human pancreatic acini did not respond to any dose of CCK or secretin. Amylase release was stimulated by carbamylcholine and neuromedin C dose-dependently and was inhibited by respective antagonists. The localizations of CCK receptors in the human duodenum were determined. High concentrations of CCK-A receptors were detected in the mucosa as well as in smooth muscle of the duodenum by microautoradiography., Conclusion: In conclusion, human pancreatic acinar cells are responsible for carbamylcholine and neuromedin C but not for secretin. Neither CCK-A nor CCK-B receptor mediates amylase release from human pancreatic acini in vitro. Pancreatic secretion in humans in vivo may be regulated indirectly by CCK (via CCK-A receptors).