Akira Kono

Lack of satiety effect of cholecystokinin (CCK) in a new rat model not expressing the CCK-A receptor gene

This work expands recent observations that Otsuka Long-Evans Tokushima Fatty (OLETF) rats show little or no pancreatic expression of the cholecystokinin (CCK)-A receptor gene. We examined whether the CCK-A and -B receptor genes were expressed in the brain (hypothalamus) of OLETF rats in comparison with control (Long-Evans Tokushima Otsuka = LETO) rats. CCK-A receptor mRNA was detected in the hypothalamus of LETO rats but not OLETF rats. The CCK-B receptor gene was expressed in the hypothalamus in both strains. Cerebroventricular administration of CCK-8 sulfate inhibited daily food intake in LETO rats, but not in OLETF rats. These results show that in OLETF rats the absence of CCK-A receptor gene expression in the hypothalamus results in hyperphagia because of lack of satiety.

Gene structure of human cholecystokinin (CCK) type-A receptor: body fat content is related to CCK type-A receptor gene promoter polymorphism

The transcriptional start site of the human cholecystokinin (CCK)‐A receptor gene was determined by the Capsite Hunting method. Two sequence changes were detected, a G to T change in nucleotide −128, and an A to G change in nucleotide −81. The homozygote (T/T, G/G) was detected in 25 of 1296 individuals (1.9%) in the cohort study. This polymorphism showed a significantly higher percent body fat and higher levels of serum insulin and leptin, compared with wild type and heterozygotes. Our study provided the possibility that polymorphism in the promoter region of the CCK‐A receptor gene may be one of genetic factors affecting fat deposition.

Role of CCK-A Receptor for Pancreatic Function in Mice : A Study in CCK-A Receptor Knockout Mice

Introduction The cholecystokinin (CCK) family of peptides and receptors is present throughout the brain and gastrointestinal tract. The CCK receptors can be pharmacologically subdivided into two subtypes: CCK-A and CCK-B. CCK-A receptor is enriched in the pancreas of mice. Aims To determine pancreatic functions in a CCK-A receptor deficient mouse mutant generated by gene targeting in embryonic stem cells. The targeting vector contained lacZ and neo insertions in exon 2. Methodology To examine exocrine functions, amylase release from the dispersed acini in vitro was examined. In the in vivo study, the mixture of bile–pancreatic juice was collected, and amylase, bicarbonate, and bile acid outputs were determined after the administration of various stimulants. The cystic duct of the gallbladder and the pylorus were ligated to exclude the involvement of gallbladder contraction and gastric acid. Pancreatic enzyme content was measured, and histologic examinations by HE and lacZ staining were conducted. To examine endocrine functions, oral glucose tolerance test (2 g/kg) was determined. Results The body weight, pancreatic wet weight, and enzyme content in the pancreas were similar among the three genotypes. Amylase release in vivo and in vitro and bicarbonate secretion in vivo were not stimulated by CCK-8 in CCK-AR (−/−) mice, whereas the responses to other stimulants were substantial in (−/−) mice. Administration of secretin did not increase bicarbonate secretion regardless of genotype. A normal glucose tolerance was observed in (−/−) mice. Acinar cells, islets, and duct cells were stained by lacZ, and HE staining revealed no pathologic findings. Conclusion The CCK-A receptor is important for pancreatic exocrine secretion, but not essential for maintaining glucose concentration and pancreatic growth in mice.

Antitumor Effect of DX‐8951, a Novel Camptothecin Analog, on Human Pancreatic Tumor Cells and Their CPT‐11‐resistant Variants Cultured in vitro and Xenografted into Nude Mice

DX‐8951 is a novel water‐soluble derivative of camptothecin. We evaluated the effects of DX‐8951 on the growth of several pancreatic tumor cell lines in vitro and in vivo. In vitro cytotoxic activity of DX‐8951 against SUIT‐2 and KP‐1N cells, as indicated by IC50 value, was several times more potent than that of SN‐38, an active metabolite of CPT‐11, and dozens of times more potent than that of SK&F104864 (topotecan). DX‐8951 also showed the greatest cytotoxicity against CPT‐11‐resis‐tant variants, SUIT‐2/CPT‐11 and KP‐1N/CPT‐11 cells, and the cross‐resistance of these cells to DX‐8951 was lower than that to SN‐38 and SK&F104864. Topoisomerase 1 inhibitory activity of DX‐8951 was about three‐fold stronger than that of SN‐38, as measured in crude nuclear extract obtained from SUIT‐2 cells. DX‐8951 induced DNA fragmentation, a specific feature of apoptosis, in SUIT‐2 cells more effectively than SN‐38. DX‐8951 exhibited potent antitumor effects against SUIT‐2 in a solid tumor model and in a liver metastasis model, in which tumor cells were xenografted sub‐cutaneously and intrasplenically, respectively, into nude mice. The in vivo effects were closely similar to or somewhat superior to those of CPT‐11, DX‐8951 also showed significant antitumor effects against SUIT‐2/CPT‐11 solid tumors, against which CPT‐11 had no effect. These results suggest that, on the basis of its strong antitumor activity and effectiveness against CPT‐11‐resistant tumors, DX‐8951 may be a useful therapeutic agent in the treatment of human cancer. The potent cytotoxicity of DX‐8951 may result from strong inhibition of topoisomerase I, which may then trigger apoptotic cell death.

Overexpression of Cholecystokinin-B/ Gastrin Receptor Gene in the Stomach of Naturally Occurring Cholecystokinin-A Receptor Gene Knockout Rats

We examined the cholecystokinin (CCK)-B/gastrin receptor, H+/K+-ATPase and somatostatin gene expression, the histology and immunohistochemistry of gastrin and somatostatin of the stomach, plasma gastrin levels, and gastric acid secretion in naturally occurring CCK-A receptor gene knockout (Otsuka Long-Evans Tokushima fatty, OLETF) rats. The CCK-B/gastrin receptor, H+/K+-ATPase and somatostatin mRNAs were determined by Northern transfer analysis. The gastric acid secretion and the plasma gastrin level were measured in vivo. The levels of CCK-B/gastrin receptor mRNA in the forestomach and the glandular stomach in OLETF rats were 2-fold higher than those of control rats, although those of H+/K+-ATPase and somatostatin mRNAs were not different. Histological examination revealed thickening of the fundic mucosa, and hyperplasia and hypertrophy of parietal cells, although immunohistochemistry of gastrin and somatostatin revealed no significant difference from the control rats. Gastric acid secretion stimulated by gastrin or histamine was enhanced, whereas the fasting plasma gastrin level was not significantly different from that in control rats. The overexpression of CCK-B/gastrin receptor mRNA and the hyperfunction of parietal cells were observed in rats without CCK-A receptor gene expression.

Differential expression of DNA topoisomerase I gene between CPT-11 acquired- and native-resistant human pancreatic tumor cell lines: detected by RNA/PCR-based quantitation assay.

RNA/PCR quantitation method was developed to determine DNA Topoisomerase I(Topo I)-specific mRNA in order to study its gene expression in CPT-11 sensitive, acquired- or native-resistant human pancreatic tumor cell lines. The results were supported by Northern blotting and Western blotting analyses. Acquired-resistant cells have shown decreased levels of Topo I mRNA, compared with their parental cells. On the contrary, in the wild type cells no correlation was shown between sensitivity and gene expression. On the other, specific Topo I activity of the native resistant cell lines was fairly lower than that of sensitive cell lines, suggesting that immunoreactive Topo I protein contains low levels of active form enzyme which could be targets of CPT-11 in these native-resistant ones. Finally, the different mechanisms might be operative between acquired- and native-resistant tumor cells.

Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.

Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1-5 fmol/injection (S/N = 3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C18 column before conducting the labelling reaction. Pro-Hyp, Pro-Gly and Pro-Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5-4.8 and 1.7-5.8%, respectively. The concentrations of Pro-Hyp, Pro-Gly, Pro-Pro, Pro and Hyp in normal human urine were 97.6 +/- 28.2, 2.74 +/- 1.48, 2.08 +/- 1.13, 6.71 +/- 3.34 and 2.30 +/- 1.59 nmol/mg creatinine, respectively.

Pancreatic Endocrine Dysfunction in Rats Not Expressing the Cholecystokinin-a Receptor

Summary Cholecystokinin (CCK) has been suggested to modulate insulin output. We have shown that Otsuka Long-Evans Tokushima Fatty (OLETF) rats show little or no expression of the CCK-A receptor gene in the pancreas. We examined whether the CCK-A and CCK-B receptor genes are expressed in the islets and the role of CCK-A receptor in insulin secretion. Gene expressions of CCK receptors were determined by the reverse-transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot hybridization and Northern transfer analysis using LETO rats as controls. Pancreatic endocrine function was examined in perfusion (exogenous CCK stimulation) and meal ingestion (endogenous CCK stimulation) studies. CCK-A receptor mRNA was detected in the islets of LETO rats but not OLETF rats. Expression of the CCK-B receptor gene was detected in both strains by RT-PCR. Insulin secretion was impaired in OLETF rats, but the insulin contents of OLETF and LETO rats were not different. No abnormalities were detected histologically in either strain. These results suggest that the occurrence of pancreatic endocrine dysfunction in OLETF rats may be due to a defect in expression of the CCK-A receptor gene, not to insulin deficiency.

Disruption of cholecystokinin (CCK)-B receptor gene did not modify bile or pancreatic secretion or pancreatic growth : A study in CCK-B receptor gene knockout mice

Pancreatic exocrine function and bile secretion were examined in cholecystokinin (CCK)-B receptor gene-targeted mice and compared among different genotypes [i.e., CCK-B receptor gene: (+/+), wild-type; (+/-), heterozygous; and (-/-), homozygous deficient]. The histology and protein concentrations in the pancreas also were examined. Amylase release from the dispersed acini was examined in vitro by using the various doses of CCK-8, carbachol, and secretin. In vivo, the bile and pancreatic juice were collected, and the concentrations of amylase and bile acid were measured in anesthetized mice. The responses to CCK (100 pmol/kg) or acetyl-beta-methylcholine (500 nmol/kg) were examined. In vitro studies showed that the maximal effective concentrations of CCK-8 (10(-l0) M), carbachol (10(-5) M), and secretin (5 x 10(-7) M) were comparable for all genotypes. Fluid, amylase, and bile acid outputs in vivo also were comparable for all genotypes. Pancreatic wet weight and protein concentrations were not significantly different, and no abnormal findings were observed on histologic examination in any genotype. These results indicated that the CCK-B receptor has no role in pancreatic growth, exocrine secretion, or bile secretion in adult mice.