[Show abstract][Hide abstract] ABSTRACT: A protein with pancreastatin-like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal residue analysis, in conjunction with the use of antibodies that are specific for the C-terminal amide peptide of porcine pancreastatin, identified this protein as a 186-amino-acid protein corresponding to human chromogranin A-116-301 (the fragment corresponding to the positions from 116 to 301 of human chromogranin A). Digestion of this protein with trypsin yielded a 48-amino-acid peptide with the retention of full pancreastatin activity. Serum from patient with insulinoma contains a peptide specie(s) that comigrates with the 48-amino-acid pancreastatin, suggesting that this peptide might be a physiologically important circulation form of pancreastatin in humans.A sensitive radioimmunoassay was established using antibody developed against a synthetic 29-amino-acid peptide amide of pancreastatin. Immunocytochemical staining revealed that a major population of human pancreatic islet cells were immunoreactive to the antiserum but with varying intensity of staining. Pancreastatin-like immunoreactivity was not observed in exocrine acinar cells.
European Journal of Biochemistry. 03/2005; 191(1):33 - 39.
[Show abstract][Hide abstract] ABSTRACT: Luminal cholecystokinin-releasing factor (LCRF), purified from rat intestinal secretions, is an intraluminal regulator of cholecystokinin (CCK) secretion during bile and pancreatic juice diversion.
Because the LCRF content was not influenced by intravenous administration of atropine or somatostatin, the inhibitory effect of a potent somatostatin analog octreotide on LCRF content, the plasma CCK level, and pancreatic secretion was examined.
Rats were prepared with bile and pancreatic cannulae and two duodenal cannulae and with an external jugular vein cannula. After 1.5-hour basal collection, bile and pancreatic juice was diverted for 2 hours, during which octreotide was infused intravenously or into the duodenal lumen. The changes in pancreatic secretion were recorded for 2 hours, and the plasma CCK level and LCRF content 2 hours after the treatment were measured.
Bile and pancreatic juice diversion significantly increased pancreatic secretion and plasma CCK and LCRF levels. Intravenous infusion of octreotide (0.2 and 0.5 nmol/kg/hour) inhibited all parameters. Intraduodenal infusion of a lower dose of octreotide (33 nmol/kg/hour) inhibited pancreatic secretion, but did not inhibit CCK release or LCRF content. The higher doses (100 and 300 nmol/kg/hour) inhibited all parameters.
Intravenous and intraduodenal administrations of octreotide inhibited CCK release and LCRF content and pancreatic secretion induced by bile and pancreatic juice diversion.
[Show abstract][Hide abstract] ABSTRACT: Exclusion of bile-pancreatic juice from the intestine increases pancreatic secretion via cholecystokinin (CCK) release in conscious rats. Luminal CCK-releasing factor (LCRF), purified from rat intestinal secretions, is an intraluminal regulator of CCK secretion during bile-pancreatic juice diversion.
Because somatostatin is a potent inhibitor of CCK release and pancreatic secretion, the inhibitory effect of somatostatin on LCRF was examined.
Rats were prepared with bile and pancreatic cannulae and two duodenal cannulae and with an external jugular vein cannula. The experiments were conducted without anesthesia. After 1.5-hour basal collection of pancreatic juice with bile-pancreatic juice return, bile-pancreatic juice was diverted for 2 hours, during which time somatostatin (2, 10 nmol/kg/h) was infused intravenously. The rats were killed before and 1 and 2 hours after bile-pancreatic juice diversion. To examine the effect of luminal somatostatin, 50 or 200 nmol/kg/h of somatostatin was infused into the duodenum. The plasma CCK and luminal content of LCRF were measured by specific radioimmunoassays.
Bile-pancreatic juice diversion significantly increased pancreatic secretion, plasma CCK, and LCRF levels. Intravenous infusion of somatostatin inhibited CCK release and pancreatic secretion, but not LCRF content. Luminal administration of somatostatin did not show any effect.
Inhibitory effect of circulating somatostatin on CCK release and pancreatic secretion is independent of LCRF content.
[Show abstract][Hide abstract] ABSTRACT: The purpose of the present study was to evaluate plasma glucagon-like peptide-1 (GLP-1) responses after oral glucose ingestion in patients with chronic pancreatitis and to clarify how GLP-1 secretion relates to pancreatic diabetes.
An oral glucose tolerance test (OGTT) was performed in 17 patients with chronic pancreatitis. Plasma glucose, immunoreactive insulin (IRI), C-peptide, glucagon, and GLP-1 levels at each time point during OGTT were measured. The diagnosis of chronic pancreatitis was made by the findings of endoscopic retrograde pancreatography (ERP): evident dilation of the main pancreatic duct with or without pancreatolithiasis.
The patients were divided into three groups according to the World Health Organization classification of diabetes based on plasma glucose levels after OGTT. The groups were: normal (three patients), impaired glucose tolerant (IGT) (six patients), and diabetic (DM) (eight patients). In the DM group, IRI and C-peptide response levels after oral glucose ingestion were significantly reduced as compared with those of the normal and IGT groups. No significant glucagon responses to oral glucose ingestion were found in the three groups. In contrast, plasma GLP-1 levels were significantly elevated after oral glucose ingestion in the DM groups as compared with normal and IGT groups.
The present study affords evidence that plasma GLP-1 levels become elevated with development of pancreatic diabetes, although the precise mechanism of this elevation remains undetermined.
The American Journal of Gastroenterology 05/1999; 94(4):976-81. · 9.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The changes in levels of the newly discovered luminal CCK-releasing factor (LCRF) in the small intestinal lumen before and after bile-pancreatic juice diversion in conscious rats were examined by a specific RIA. Moreover, we also examined whether LCRF secretion was under cholinergic control. Anti-LCRF antiserum was raised in rabbits, and a sensitive RIA was established. The localization of LCRF was examined by immunohistochemistry. The luminal content of LCRF was significantly increased by bile-pancreatic juice diversion, during which luminal trypsin activity was eliminated. The increase in luminal LCRF content was not inhibited by intravenous infusion of atropine. The changes in plasma levels of CCK and pancreatic secretion were similar to those in luminal LCRF contents. LCRF immunostaining was observed in villus tip enterocytes of the small intestine and was most prominent in the duodenal portion. These results support our original hypothesis that LCRF may be released spontaneously into the small intestinal lumen from the villus tip enterocytes and its intraluminal degradation by proteases regulates CCK release. Furthermore, LCRF release was not subject to cholinergic regulation.
The American journal of physiology 02/1999; 276(1 Pt 1):G287-92. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antisecretory factor (AF) was identified as a pituitary protein that inhibits the intestinal fluid secretion induced by cholera toxin. One aim of this study was to elucidate whether AF is also synthesized in the intestine or if AF produced in the pituitary is transported to the intestinal tract for its function there. cDNA clones encoding a protein proposed to be AF were isolated from rat pituitary gland and intestinal mucosa cDNA libraries. The nucleotide sequences of clones isolated from the rat pituitary gland and intestinal mucosa were identical. The deduced amino acid sequence was highly homologous to the sequence for subunit 5a of the human 26S protease that exists abundantly in the cytosol and nucleus. The production of AF in the intestine was confirmed by Northern blot and immunoblot analyses. Immunocytochemical observations of cells transfected with the rat AF cDNA showed that the AF protein was localized in the cytoplasm. These findings suggest that the protein proposed to be AF may be a cytoplasmic protein, it exists in the intestine rather than being transported from the pituitary gland, and it may function in intestinal cells.
Biochemistry and Cell Biology 02/1999; 77(3):223-8. · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growth of the human pancreatic carcinoma cell line KP-1N was stimulated with cholecystokinin (CCK)-8. A 40% increase in cell numbers was observed in the presence of 10(-10) MCCK-8 and this increase was inhibited by the addition of 25 microM CCK-A receptor antagonist (CR1505). The binding affinity of CCK-8 to KP-1N cells was 21-fold higher than that of gastrin 17-I. No significant increase in intracellular Ca2+ concentration was found upon stimulation with CCK-8. Components of signal transduction pathways that were activated in KP-1N cells after stimulation with CCK-8 were studied. CCK-8 stimulated tyrosine phosphorylation of a mitogen-activated protein kinase (MAPK) of approximately 42 kDa (p42map). c-Jun amino-terminal kinases (JNKs) of 46 kDa (p46jnk) and 55 kDa (p55jnk) were also activated by CCK-8 and increased the phosphorylation of c-Jun. CCK-8 at 10(-7) M induced 1.5-fold increases in the phosphorylation of MAPK and of c-Jun by JNKs, respectively. These results suggest that cell proliferation stimulated with CCK-8 in KP-1N cells may be mediated by signal transduction cascades leading to activation of JNKs and MAPKs.
[Show abstract][Hide abstract] ABSTRACT: We examined the role of the cholecystokinin-A (CCK-A) receptor in acute inflammatory and regenerative stages of experimental pancreatitis using a rat model lacking the CCK-A receptor [Otsuka Long-Evans Tokushima Fatty (OLETF) rats]. OLETF and control [Long-Evans Tokushima Otsuka (LETO)] rats were prepared with an internal bile fistula and with obstruction of pancreatic flow and were sacrificed 1-14 days later. Histological examination was performed, and changes in pancreatic wet weight, protein concentration, CCK-A and -B receptor mRNA levels, tyrosine kinase activities, and plasma amylase and CCK levels were determined. The plasma amylase level showed a transient increase on day 1, the CCK level remained at high levels throughout, and tyrosine kinase activity was increased significantly on day 3 but declined thereafter. These parameters were comparable for both strains during the acute inflammatory stage. However, no regenerative findings were observed by histological examination and the protein concentration in the pancreas was significantly lower in OLETF rats on days 7-14, during which time regeneration was completed in LETO rats. These observations indicate that the absence of the CCK-A receptor did not modify the acute phase of pancreatitis but significantly retarded regeneration of the pancreatic tissue.
[Show abstract][Hide abstract] ABSTRACT: To clarify the mechanism(s) of the antidiabetic effects of truncated glucagon-like peptide-1 (GLP-1) in diabetics, we examined its insulinotropic and extrapancreatic effects in a newly established strain of spontaneously non-insulin-dependent diabetic (NIDDM) rats, Otsuka Long-Evans Tokushima Fatty (OLETF) rats, that received a continuous infusion of truncated GLP-1 620 pmol/d/kg (G group, n = 12) or of vehicle (V group, n = 12) for 4 weeks by Alzet pump. Nonfasting plasma glucose levels were significantly lower (P < .05) in the G group than in the V group (7.0 +/- 0.67 v 9.1 +/- 1.7 mmol/L), and fasting plasma immunoreactive insulin (IRI) levels were lower in the former than in the latter (0.63 +/- 0.31 v 0.78 +/- 0.25 nmol/L). At day 15 of infusion, the G group showed an attenuated plasma glucose response to an oral glucose load, but had plasma IRI levels comparable to those in the V group. A long-term infusion of truncated GLP-1 increased the glucose infusion rate (GIR) significantly (P < .05) during a euglycemic-hyperinsulinemic clamp test (59.0 +/- 14.8 mumol/kg/min for group G v 38.9 +/- 12.2 for group V), but hepatic glucose output (HGO) did not differ significantly for either group. Uptake of 2-deoxy-D-glucose (2DG) by peripheral muscles in the G group was as much as 2.4-fold higher than in the V group (5.52 +/- 2.04 v 2.29 +/- 0.97 mumol/100 g muscle weight/min). We conclude from these data that truncated GLP-1, in addition to its well-known incretin effect, is capable of augmenting insulin action in peripheral tissues of diabetics, which can contribute, in part, to improve glucose intolerance in OLETF rats.
[Show abstract][Hide abstract] ABSTRACT: The immunohistochemical localization of pancreastatin (PST) was examined in brains of Alzheimer's disease (AD) and control cases using three different antisera to PST, and was compared with the staining for chromogranin A (CgA), the precursor of PST. In control brains, CgA-like immunoreactivity was observed in the cytoplasm and fibers of certain neuronal populations, which were not immunostained with any of the PST antisera. In AD brains, dystrophic neurites of globular shape located in senile plaques were immunostained with each of the PST antisera, as well as with the CgA antibody. PST-positive and CgA-positive dystrophic neurites showed similar profiles. The present study indicates that CgA is probably cleaved to produce PST in some globular dystrophic neurites in senile plaques.
[Show abstract][Hide abstract] ABSTRACT: Despite the widespread distribution of chromogranin A (CgA) in neuroendocrine tissues, the biological function of CgA has not yet been elucidated. The primary amino acid sequence of CgA, elucidated by cDNA analysis, has been revealed to include several pairs of basic amino acid residues that are homologous to the bioactive peptides, such as pancreastatin (PST) and chromostatin (CST). Using antibodies for human PST and CST, the immunohistochemical localization of these peptides was investigated in neuroendocrine tissues, including human pituitary glands, pancreas, adrenal medulla, various types of neuroendocrine neoplasms (13 pheochromocytomas, 10 medullary thyroid carcinomas, 11 pancreatic endocrine tumors, and 19 carcinoid tumors), and the cell line QGP-1N derived from human somatostatin-producing pancreatic endocrine tumor. Variable immunoreactive intensities of PST and CST were seen, but both peptides were detectable in all neuroendocrine tissues and in most of the neoplasms. Immunoreactivity for both PST and CST was observed in 100 and 73%, respectively, of pancreatic endocrine tumors, all pheochromocytomas, and 80 and 40%, respectively, of medullary thyroid carcinomas, as well as all nonrectal carcinoid tumors. In rectal carcinoids, cells immunoreactive for PST and CST were sparse. The distribution of PST and CST was similar to that of CgA, and it is considered that these peptides are simultaneously processed from CgA, and may play roles in autocrine and paracrine regulation on various hormones in addition to their previously known functions.
[Show abstract][Hide abstract] ABSTRACT: The plasma levels of chromogranin A (CGA) in patients with islet cell tumor and plasma CGA responses to administration of a somatostatin analogue (Octreotide) in two of these patients were examined in comparison with plasma pancreastatin (PST) levels. There was a significant correlation between the fasting plasma levels of CGA and PST (r = 0.6, P < 0.001). Administration of the somatostatin analogue reduced the plasma concentrations of PST and CGA within 1 h, but the responses of CGA and PST to the analogue were not parallel in either patient. Thus, the suppressive effects of the analogue on the secretions of PST and CGA may be different. The results suggest the value of the PST and CGA assays used in this study.
Life Sciences 02/1995; 57(9):889-95. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pancreastatin (PST) is processed from chromogranin A and the C-terminal amide of the peptide is an absolute requirement for biological activities. Human pancreatic carcinoma cells QGP-1 which produce both chromogranin A and PST were used to isolate cDNAs encoding two forms of peptidylglycine alpha-amidating monooxygenase (PAM). The two forms are a full length bifunctional enzyme and a variant lacking the transmembrane domain-coding region. When the cDNAs of these two forms were expressed in COS-7 cells, cells transfected with the predicted soluble form released into the culture medium a very much higher amidating activity which converts human chromogranin A-(273-302) to PST-29. The optimal pH for amidating activity was 5.4 and Cu2+, ascorbate and catalase were required as cofactors for the both forms of PAM. Km values for the membrane-bound and the soluble forms of PAM were 15.7 +/- 3.1 microM and 12.4 +/- 1.6 microM, respectively. These results demonstrate that both forms of PAM can function in the posttranslational processing of chromogranin A to PST in the environment of a secretory vesicle.
Biochemical and Biophysical Research Communications 12/1994; 205(1):282-90. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Different glucagon-like peptide-1 (GLP-1) molecular forms are produced in the pancreas and the small intestine by differential processing of proglucagon. In this report, molecular forms of GLP-1 in two human pancreatic endocrine tumors were studied and compared with those in the pancreas and small intestine. A predominant GLP-1 immunoreactive form in the pancreas was eluted at the position of GLP-1(1-36) amide, whereas a predominant immunoreactive form in the ileal mucosa was eluted at the position of GLP-1(7-36) amide. In a glucagonoma, GLP-1 immunoreactive forms corresponding to GLP-1(7-36) amide and GLP-1(7-37) were predominant and immunoreactive forms at the position of GLP-1(1-36) amide and GLP-1(1-37) were minor. In another tumor, an insulinoma, immunoreactive forms were detected at the positions of GLP-1(7-36) amide, GLP-1(7-37), GLP-1(1-36) amide and GLP-1(1-37). Thus, the pattern of GLP-1 molecules in pancreatic tumors was not a pancreatic pattern and mimicked that found in the small intestine or consisted of both the patterns found in the small intestine and the pancreas. These data suggest that neoplastic transformation of the islet cells is associated with a switching in processing phenotype from islet (A) cells to intestinal (L) cells.
Diabetes Research and Clinical Practice 09/1994; 25(1):43-9. · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromogranin A (CGA) is thought to be a precursor of pancreastatin (PST). Carbachol (Cch) stimulated the secretion of CGA and PST from QGP-1N cells derived from a human pancreatic islet cell tumor. Atropine inhibited the secretion of both. Sodium fluoride, phorbol ester, and calcium ionophore also stimulated the secretion of both. Cch (10(-5) M) stimulated inositol 1,4,5-trisphosphate production in QGP-1N cells. Stimulation with Cch increased the total amount of PST in the cells and the medium 1.7-fold and decreased the amount of CGA in the cells and medium. QGP-1N cells were labelled with [35S]methionine, and then CGA and PST in the cells and medium were immunoprecipitated with specific antisera, and separated by electrophoresis in polyacrylamide gel. Stimulation with Cch resulted in an increase in the intensity of PST-immunoreactive bands and a decrease in those of CGA-immunoreactive bands. Cch did not increase the cellular level of CGA messenger RNA. These results suggested that (1) the secretion of CGA and PST from QGP-1N cells is regulated mainly through muscarinic receptors coupled with activation of polyphosphoinositide breakdown by a G protein, with intracellular calcium ion and protein kinase C playing a role in the stimulus-secretion coupling and that (2) Cch may induce the secretion of PST and CGA and processing from CGA to PST.
[Show abstract][Hide abstract] ABSTRACT: The concentrations of pancreastatin-like immunoreactivity (PST-LI) of the cerebrospinal fluid (CSF) were measured in the patients with Alzheimer type dementia (ATD) and in age-matched normal subjects. The mean PST-LI concentration in the CSF of ATD patients was significantly lower than that of normal subjects. Gel chromatographic analysis revealed that the main PST-LI peak of ATD's CSF eluted at molecular weight (MW) 13.5 kDa. However, the age-related change of the molecular forms of PST-LI in CSF was observed in normal subjects as following; PST-LI in neonatal CSF showed one peak at MW 13.5 kDa, that of 16–64-year-old showed two peaks at MW 13.5 and 5.4 kDa, however, only one main peak was shown at MW 5.4 kDa in the CSFs of 72–85-year-old. These findings suggest that the production of PST-LI was decreased and the proteolytic cleavage, which should process big PST to PST (1–52) in normal subjects, was altered to that of neonatal type in the CNS of the patients with ATD.
[Show abstract][Hide abstract] ABSTRACT: Cholecystokinin, a brain gut peptide that stimulates gall bladder contraction and pancreatic exocrine secretion, also acts as a neurotransmitter. In this study, we demonstrated that small amounts of cholecystokinin precursor mRNA were expressed in the heart, lung, and kidney, as well as in the brain and the small intestine. The nucleotide sequences of the coding regions of the cholecystokinin precursor mRNA in these tissues were identical to those of the small intestine, indicating that cholecystokinin precursor proteins produced in these tissues are identical to those in small intestine. This is the first report demonstrating that the cholecystokinin precursor gene is expressed in the heart, lung, and kidney, as well as in the gastrointestinal tract and brain.
Journal of Gastroenterology 05/1994; 29(2):125-8. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using a specific antiserum for the C-terminal glycine amide region of human pancreastatin (PST), pancreastatin-like immunoreactivity (PST-LI) was measured in cerebrospinal fluid (CSF) from 447 subjects (368 ± 10.8 pmol/l, mean ± S.E.M.) free from endocrine diseases. The CSF contents of PST-LI showed a mountain-shape type change which peaked at 40 years of age. The highest concentration was found in the group of ages 40–49 years old (412 ± 22.9 pmol/l) and the lowest concentration was found in the group of ages 80–89 years old (293.2 ± 45.2 pmol/l) among various age groups. Gel chromatographic examination revealed the presence of two major forms (MW 13,500 and 5,400) of PST-LI in CSF. Because of the character of this antibody, the large molecular form is possibly an N-terminally elongated PST and the other may be PST-52. This may be the first report on the unique age-related change of PST concentration in CSF.