Catfish Pancreas Research Articles

Unique localization of disseminated pancreas in the oesophagus of catfish (clarias gariepinus) with reference to sexual dimorphism.

The fish pancreas has been reported to be composed of two portions: compact and disseminated. However, little has been elucidated in catfish. The present study describes a unique localization of the disseminated pancreas in African catfish. The sections were obtained and used for either routine histological examination following staining with haematoxylin and eosin (H & E), periodic acid-Schiff's, or were subjected to immunohistochemical staining for detection of both insulin-producing β cells and glucagon-producing alpha cells. Our investigation showed that the pancreas of catfish consisted of both compact and disseminated portions. The compact pancreas was embedded in the mesenteric adipose tissue between the spleen, stomach and liver. However, the disseminated one showed unique localization in the tunica adventitia of the middle portion of the oesophagus. The pancreas consisted of two portions, exocrine and endocrine. Furthermore, in both types of pancreas, the female showed a significantly higher ratio for the endocrine islet area/pancreatic tissue area than that of the male and also a significantly higher ratio for both insulin- and glucagon-positive area/islet area in the female pancreas (compact and disseminated) than that of the male. The present study provides evidence on a unique localization of the disseminated pancreas in the oesophagus of catfish. Furthermore, we revealed sex-related difference in the endocrine portion in both pancreatic tissues with more development in the female. The study suggests that sex hormones could be contributed to such sexual dimorphism. However, further investigation is required to compare the degree of development during the spawning and resting seasons.

Immunohistochemical distribution of glucagon-like peptide 1 in the digestive system of different aquatic vertebrates.

The distribution of glucagon-like peptide 1 (GLP-1)-positive cells in digestive tracts and pancreases of aquatic vertebrates was investigated by immunohistochemical staining method. The results suggested that GLP-1-positive cells were distributed in the columnar mucous epithelium and tubular glands of lamina propria in the digestive system. However, GLP-1-positive cells were also found in subepithelial lamina propria of the mucosae and muscularis in each segment of the digestive tract of Rana nigromaculata. The distribution densities of these cells reached peaks in the stomachs, and the middle or end segments of small intestines of Chinese softshell turtle, Bufo gargarizans, R.nigromaculata and catfish, and there was the third distribution density peak in the rectum of catfish. The total amount or overall density of GLP-1-positive cells varied a lot in the digestive tracts of different animal species. The distribution density was relatively low in the digestive tract of chub and reached the maximum in the digestive tracts of snakehead and catfish, but no GLP-1-positive cells were found in the digestive tract of bighead carp. GLP-1-positive cells were densely distributed in the pancreases of Chinese softshell turtle, B.gargarizans and R.nigromaculata. These cells spread over the superficial layers of islets or scattered in exocrine pancreas in the pancreas of B.gargarizans, spread in the endocrine cells or scattered in the pancreas of Chinese softshell turtle, scattered in the pancreas of R.nigromaculata and distributed in the superficial layers of islets in the pancreas of catfish.

Purification and properties of serine proteinases from European catfish Silurus glanis L. pancreas

Three trypsin isoforms (designated as T1, T2, and T3), three chymotrypsin isoforms (Kh1, Kh2, and Kh3), and two elastase isoforms (E1 and E2) were isolated from the pancreas of European catfish Silurus glanis L. by salting out with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion exchange chromatography on DEAE cellulose. Isoelectric points of the enzymes, determined by isoelectric focusing, amounted to 4.42 for T1, 5.64 for T2, 6.90 for T3, 4.93 for Khl, 5.23 for Kh2, 6.18 for Kh3, 6.17 for E1, and 8.48 for E2. Molecular weights of proteinases within each group were close and amounted to 30100 Da for trypsins, 39800 Da for chymotrypsins, and 24000 Da for elastases. The enzymes isolated displayed maximal activities at alkaline pH values. Inhibitor analysis demonstrated that all the proteinases isolated from European catfish pancreas belonged to the serine type.

2-Deoxyglucose stimulates the release of insulin and somatostatin from the perfused catfish pancreas

Glucokinase was proposed to function as a glucose sensor in pancreatic B-cells, acting possibly as a pacemaker of the rate of glycolysis. Glucose, mannose, and 2-deoxyglucose are good substrates of glucokinase which are easily taken up into B-cells. Glucose and mannose are well-known stimuli of insulin release in mammals and fish. I report here that 2-deoxyglucose is also a strong stimulus of insulin and somatostatin release from the in vitro perfused pancreas (i.e., splenic Brockmann body) of channel catfish ( Ictalurus punctatus). This is surprising because the product of the glucokinase-catalyzed phosphorylation of 2-deoxyglucose, 2-deoxyglucose-6-phosphate, cannot be metabolized further at an appreciable rate. 3-O-Methylglucose, which does not bind appreciably to mammalian glucokinase, stimulated neither insulin nor somatostatin release. Glucosamine, which binds tightly to glucokinase but is phosphorylated only at a very low rate, did not stimulate insulin release either, but did cause a small amount of somatostatin to be released. The results suggest that glucose-activated glucokinase itself may serve as a signal molecule in glucose recognition by B- and D-cells.

Identification of disulfide-containing peptides in endocrine tissue extracts by HPLC-electrochemical detection and mass spectrometry

A new procedure to selectively identify disulfide-containing peptides in extracts of biological tissues is described. Disulfide-containing peptides are detected by their UV absorbance and electrochemical (EC) activity after chromatographic separation, and subsequently identified by fast atom bombardment mass spectrometry (FABMS). This combination of fractionation by HPLC and selective detection is attractive because it is rapid, highly specific for disulfide-containing peptides, and applicable to all disulfide-containing peptides that may be present in complex biological mixtures. Useful procedures for applying the method are demonstrated with tissue extracts from bovine pituitary and catfish pancreas. In addition to finding the expected disulfide-containing peptides, evidence for two forms of catfish insulin are presented. The merits of this and other methods used to detect peptides in similar tissue extracts are discussed.

The use of plasma desorption time-of-flight mass spectrometry to screen for products of prohormone processing in crude tissue extracts

Californium-252 plasma desorption mass spectrometry (252Cf PDMS) of a crude, desalted, extract of piscine endocrine pancreas provided mass information for the major biologically active peptide hormones present in this tissue. An extraction procedure compatible with 252Cf PDMS analysis was developed. In extracts of catfish pancreas, strong molecular ions were identified in the positive mode for somatostatin-14 (1638 amu), O-glycosylated somatostatin-22 (2944 amu), glucagon (3512 amu), glucagon-like peptide (3785 amu), insulin (ca. 5550 amu), and other prohormone-derived peptides. Both protonated species and sodium adducts were apparent in the mass spectrum. A number of other molecular ions were observed including somatostatin-26, 1–10 (1014 amu) and the entire portion of prosomatostatin-22 remaining after removal of somatostatin-22 (6465 amu). The data obtained by this method also resulted in the identification of the third major product of proglucagon processing in catfish pancreas, glicentin-related polypeptide. Subtractive Edman degradation analyzed by 252Cf PDMS was also used to confirm a mass assignment.

Purification and characterization of a collagenolytic serine proteinase from the catfish pancreas.

A collagenolytic enzyme was purified to homogeneity from the activated pancreatic extract of the catfish Parasilurus asotus by chromatography on DEAE-cellulose, hydroxylapatite, and Cellulofine columns. The molecular weight of the enzyme was estimated to be 29,500 by SDS-polyacrylamide gel electrophoresis. The enzyme is capable of degrading native, reconstituted calf skin collagen fibrils at pH 7.5 and 37 degrees C, and also of reducing the viscosity of native calf skin collagen at pH 7.5 and 20 degrees C. The SDS-polyacrylamide gel electrophoresis of thermally denatured enzyme-collagen reaction mixtures showed that the enzyme can cleave peptide bonds in the non-helical and triple-helical regions of the collagen. The enzyme was inhibited by DFP, PMSF, soybean trypsin inhibitor, and chicken ovoinhibitor, but not by metal-chelating reagents EDTA, EGTA, o-phenanthroline, or L-cysteine. These results indicate that the enzyme is a unique collagenolytic proteinase belonging to the group of serine proteinases and is a new member of the class of pancreatic enzymes.

Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas.

Both glucagon and the structurally similar glucagon-like peptide proteolytically derived from preproglucagon were purified from the endocrine pancreas of the channel catfish (Ictalurus punctata). This study represents the first report of the isolation of glucagon-like peptide from any source. Peptide sequences of glucagon-like peptide from other species have only been deduced from the cDNA sequences for preproglucagon. The sequence of the 34-residue glucagon-like peptide was found to be HADGTYTSDVSSYLQDQAAKDFITWLKSGQPKPE. Catfish glucagon-like peptide shares sequence identity at 26 of 31 residues with the putative glucagon-like peptide from anglerfish preproglucagon II. The mass of catfish glucagon-like peptide was found by fast atom bombardment-mass spectrometry to be 3785, identical with the value predicted by sequence analysis. This suggests that no post-translational modification occurs beyond proteolytic processing. The sequence of catfish glucagon was determined to be HSEGTFSNDYSKYLETRRAQDFVQWLM(N,S). Catfish glucagon exhibits a high degree of immunologic similarity with porcine glucagon by radioimmunoassay, whereas catfish glucagon-like peptide does not.

Studies on pancreatic enzymes in fish. XIV. Purification and some properties of carboxypeptidase A from the catfish pancreas.

Studies on pancreatic enzymes in fish. XIV. Purification and some properties of carboxypeptidase A from the catfish pancreas.

Studies on pancreatic enzymes in fish. XV. Enzymic characterization of carboxypeptidase A from the catfish pancreas.

Studies on pancreatic enzymes in fish. XV. Enzymic characterization of carboxypeptidase A from the catfish pancreas.

Difference in glucose dependency of insulin and somatostatin release.

The splenic Brockmann body of channel catfish (Ictalurus punctatus) was isolated and perfused in vitro. It has been shown previously that the hormone release from the catfish pancreas in response to various stimuli resembled that of a mammalian pancreas. The release of insulin and somatostatin was measured at various concentrations of glucose in the perfusate, and the corresponding dose-response curves were derived. As in a variety of mammalian pancreases, insulin release was stimulated half-maximally at ca. 9 mM glucose. In contrast, half-maximal somatostatin release occurred already at ca. 5 mM glucose. Although a comparable finding with mammalian D cells has not yet been reported, histological and physiological resemblances of the mammalian and the fish pancreas suggest that mammalian D cells may also have a glucose sensitivity in the 5 mM range.

Purification and characterization of a new metalloproteinase with elastolytic activity from the catfish pancreas

An elastolytic enzyme was purified to homogeneity from the pancreas of the catfish Parasilurus asotus by chromatography on CM-cellulose column and affinity chromatography on (Ala) 3-CH-Sepharose 4B column. The molecular weight of the enzyme was estimated to be 24 000 by SDS-polyacrylamide gel electrophoresis. The enzyme had elastolytic activity as well as activity toward Suc-(Ala) 3-NA. The enzyme was inhibited by EDTA, EGTA, o-phenanthroline, dithiothreitol, and cysteine, but not by the serine proteinase inhibitors iPr 2P-F and PhCH 2SO 2F. In addition, the enzyme was found to require Zn 2+ for activity. These results indicate that the catfish enzyme belongs to the group of metalloproteinases and that it is a new type of pancreatic enzyme, unlike the pancreatic elastases which are serine proteinases.

Studies on pancreatic enzymes in fish. X. Enzymic characterization of two carboxypeptidases B from the catfish pancreas.

Two carboxypeptidases B from the pzncreas of the catfish Parasilurus asotus were found to have pH optima of 7.5 and were found to be stable between pH 6.5 and 9.0. Both enzymes were strongly inhibited by EDTA, o-phenanthroline, and cysteine. The enzymes inactivated by EDTA were regenerated almost completely by the addition of ZnCl2. These results indicate that both enzymes are metalloenzymes which require zinc for activity. Both enzymes were found to be able to hydrolyze Bz-Gly-Arg and Bz-Gly-Lys. These results indicate the close similarity between two catfish carboxypeptidases B and suggest that the catfish enzymes are closely related to carboxypeptidases B from other species.

Studies on pancreatic enzymes in fish. IX. Purification and some properties of two carboxypeptidases B from the catfish pancreas.

°No carboxypeptidase A activity was detected in the extract of pancreas of the cafish Parasilurus asotus. However, when the extract was left at 4C, carboxypeptidase A activity increased with the time and reached to a maximum level after 4 days. These results indicate that carboxypeptidase A is stored as zymogen in the pancreas.Carboxypeptidase A was isolated from the autoactivated extract of the catfish pancreas by CM-cellulose column chromatography and affinity chromatography. The homogeneity of the enzyme was demonstrated by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated as 34, 000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition of the catfish enzyme was found to be similar to those of the carboxypeptidases A from other species.

Studies on pancreatic enzymes in fish - IV. Purification and some properties of anionic trypsin from the catfish pancreas.

Active form of trypsin was not found in the extract of the pancreas of the catfish Parasilurus asotus. Zymogens were activated autocatalytically to maximal level when the extract was incubated at pH 8.0 and 4°C for 4 days in the presence of calcium ions. The presence of two types of trypsin, anionic and cationic forms, in the autoactivated preparation of the extract was demonstrated by DEAE-cellulose column chromatography. Sephadex G-75 gel filtration and DEAE-cellulose column rechromatography were used for furhter purification. In the present study, an anionic trypsin was purfied to homogeneity. The molecular weight of catfish anionic trypsin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to be 26, 000. The isoelectric point of this enzyme was determined to be 5.4 by isoelectric focusing. The amino acid composition of this enzyme was similar to that of anionic trypsins from some species of vertebrates, including fishes.

Purification and some properties of elastase from the pancreas of catfish.

No elastase activity was detected in the extract of the pancreas of the catfish Paracilurusasotus. However, some activity was found in the extract activated by bovine trypsin or autocatalytically. These facts indicate that elastase is stored as zymogen in the pancreas of the catfish. Two major elastases were found in the autoactivated preparation of the extract of the pancreas. One of the enzymes, designated as elastase B, was purified by CM-cellulose chromatography. The homogeneity of the purified enzyme was demonstrated by polyacrylamide gel electrophoresis in the absence or presence of sodium dodecyl sulfate. Elastase B had strong elastolytic activity as well as Suc-(Ala)8-NA-hydrolyzing activity. The optimum pH and temperature of the enzyme were found to be near pH 8.0 and 50 to 60°C, respectively. Elastase B was found to be stable between pH 5 and 9 below 60°C.

Isolation and structure of a peptide hormone predicted from a mRNA sequence. A second somatostatin from the catfish pancreas.

This laboratory recently observed a cDNA reverse transcript to a specific mRNA from the channel catfish (Ictalurus punctata) pancreas which predicted the presence of a somatostatin gene product different from the previously reported catfish somatostatin (Oyama, H., Bradshaw, R. A., Bates, O. J., and Permutt, A. (1980) J. Biol. Chem. 255, 2251-2254). The gene product was purified using reverse phase high performance liquid chromatography. The peptide was shown to be structurally identical with somatostatin-14 from other species by tryptic mapping, amino acid composition, and sequence. The level of somatostatin-14 in the catfish endocrine pancreas was determined to be 104 micrograms of immunoreactive somatostatin/g of tissue. These observations suggest that the catfish pancreas has at least two structurally distinct somatostatins.

魚類のすい臓酵素に関する研究 Ｉ ナマズのすい臓トリプシノーゲンおよびキモトリプシノーゲンの活性化

The conditions for the activation of trypsinogen and chymotrypsinogen in the extract of the pancreas of catfish Paracilurus asotus are described. The cativation of these zymogens and the stability of the activated enzymes in the extract were in vestigated with respect to temperature, time, pH, and concentrations of Ca2+ and activator (porcine enteropeptidase and bovine trypsin). The optimal conditions for zymogen activation were established: Trypsinogen was activated to maximal level by porcine enteropeptidase (100μg⁄ml) at 4°C after 2-3 days in 50 mM Tris_HCL buffe, pH 8.0, containing 50mM CaCl2. Chymotrypsinogen was activated to maximal level by bovine trypsin (20μg⁄ml) at 4°C after 2-3 days in 50 mM Tris-HCI buffer, pH 8.0, containing 50mM CaCl2. Both tryptic and chymotrytic activies observed by activation under the optimal conditions were directly related to the amounts of protein in the extract when the protein concentrations ranged from 0.4 to 3.3mg⁄ml and 0.4 to 1.6mg⁄ml, respectively. The results indicate that the potential activities of zymogens in the pancreas of catfish can be determined by using the above methods.