Rat Pancreas Tissue Research Articles

Ligands of Peroxisome Proliferator-activated Receptor-γ Block Activation of Pancreatic Stellate Cells

Activated pancreatic stellate cells (PSCs) have recently been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcription factor which controls growth, differentiation, and inflammation in different tissues. Roles of PPAR-gamma activation in PSCs are poorly characterized. Here we examined the effects of PPAR-gamma ligands on the key parameters of PSC activation. PSCs were isolated from rat pancreas tissue, and used in their culture-activated, myofibroblast-like phenotype. Activation of PPAR-gamma was induced with 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or with troglitazone. Expression of PPAR-gamma was predominantly localized in the nuclei, and PPAR-gamma was transcriptionally active after ligand stimulation. PPAR-gamma ligands inhibited platelet-derived growth factor-induced proliferation. This effect was associated with inhibition of cell cycle progression beyond the G1 phase. PPAR-gamma ligands decreased alpha-smooth muscle actin protein expression and alpha1(I) procollagen and prolyl 4-hydroxylase(alpha) mRNA levels. Activation of PPAR-gamma also resulted in the inhibition of inducible monocyte chemoattractant protein-1 expression. 15d-PGJ2, but not troglitazone, inhibited the degradation of IkappaB-alpha and consequent NF-kappaB activation. In conclusion, activation of PPAR-gamma inhibited profibrogenic and proinflammatory actions in activated PSCs, suggesting a potential application of PPAR-gamma ligands in the treatment of pancreatic fibrosis and inflammation.

Three-dimensional high voltage electron microscopy of thick biological specimens

The procedures recently developed in our laboratory to observe three-dimensional structures of cell organelles in thick biological specimens by means of high voltage electron microscopy are reviewed. Thick biological specimens such as whole mount cultured cells seeded and grown on grid meshes in culture vessels or thick sections cut from embedded tissues and stained by histochemical reactions can be readily observed three-dimensionally by high voltage transmission electron microscopy at 400–1000kV. Cultured cells used were both primary cultures from animal tissues and established cell lines maintained in our laboratory. The livers of adult Wistar rats were isolated by collagenase perfusion, and hepatocytes were suspended in a Leibovitz medium and seeded on formval coated gold grid meshes in Petri dishes, incubated in a CO2 incubator in a humidified atmosphere containing 5% CO2 in air at 37°C for a few days. Established cell lines, CHO-K1 cells, were cultured in Ham's F12 medium, while HeLa cells were cultured in Eagle's MEM under the same condition. Some of the cells were cultured under experimental conditions such as hepatocyte culture in the medium containing peroxisome proliferating agents such as clofibrate or bezafibrate and some of them were labeled with 3H-thymidine, 3H-uridine, 3H-labeled precursors and 14C-bezafibrate. Also some cells were incubated in medium containing HRP to induce pinocytosis. All the whole mount cultured cells on grid meshes were prefixed in buffered 2.5% glutaraldehyde, stained with various histochemical reactions and postfixed in 1% osmium tetroxide. The histochemical reactions used were glucose-6-phosphatase (G-6-Pase), thiamine pyrophosphatase (TPPase), cytochrome oxidase, acid phosphatase (AcPase), DAB, ZIO, PA–TCH–SP reactions and radioautography was performed after labeling with radiolabeled compounds. The whole mount cultured cells were dried in a critical point dryer and were observed with JEOL JEM-4000EX or Hitachi H-1250M high voltage electron microscopes at 400–1000kV. By tilting the specimens' stereo-pair micrographs were recorded and they were observed with stereoscopes. Rat liver, mouse intestine and pancreas tissues, fixed and stained as above, were embedded in Epoxy resin, thick sectioned at 1–2μm and were observed as for the whole mount cultured cells at 1000kV. Stereo-pairs were further analyzed with an image analyzer JEOL JIM-5000 (JEOL, Tokyo, Japan), producing two contour lines plotted from the micrographs at a thickness of 0.2μm and were observed with anaglyph type glasses, demonstrating the depth or heights of respective cell organelles. The results show that whole mount cultured cells and thick sections stained with histochemical reactions reveal cell organelles corresponding to marker enzymes, such as G-6-Pase in endoplasmic reticulum, TPPase and ZIO in Golgi apparatus, cytochrome oxidase in mitochondria, AcPase in lysosomes, DAB in peroxisomes and pinocytotic vesicles, PA–TCH–SP in secretory granules, 3H-thymidine and 3H-uridine in nuclei, 3H-animo acids in endoplasmic reticulum and secretory granules, 14C-bezafibrate around ER and peroxisomes. The ultrastructure of these cell organelles as well as the structural relationship between them can be demonstrated three-dimensionally with stereo-pair images. Overall, these procedures are useful for analyzing stereologically the ultrastructure of cell organelles in cells and tissues.

Cellular localization of calmodulin-dependent protein kinases I and II to A-cells and D-cells of the endocrine pancreas.

Ca2+/calmodulin-dependent protein kinases I and II, initially identified in brain on the basis of their ability to phosphorylate synapsin I, have been implicated in the regulation of Ca2+-dependent synaptic neurosecretion. Specific recombinant and synthetic peptide antibodies were used to examine the distribution of CaM kinases I and II in the rat pancreas and other tissues. The CaM kinase I antibodies detected a doublet of cytosolic proteins of approximately 38 and approximately 42 kD by immunoblot. CaM kinase I was observed in glucagon-containing A-cells at the periphery of the islet of Langerhans but had little or no overlap with pancreatic polypeptide or somatostatin cells. In contrast, CaM kinase II was localized to somatostatin-containing D-cells. CaM kinase I co-localized with glucagon secretory granules. CaM kinase II was not associated with the somatostatin granule but rather was enriched in areas of the cells that contained relatively little somatostatin. Because glucagon secretion is Ca2+-dependent, it is attractive to speculate that CaM kinase I may play a regulatory role in glucagon secretion. Glucagon and somatostatin cells both utilize intracellular Ca2+ for signaling. Therefore, specific CaM kinases may act as effectors of Ca2+ in these different cell types.

Identification of rat pancreatic duct cells by their expression of cytokeratins 7, 19, and 20 in vivo and after isolation and culture.

Cells from the excretory ducts of the pancreas are thought to be capable of differentiating into exocrine and endocrine cells. To study this in rat models, markers must be found to identify the cells under different experimental conditions. We tested antibodies to different cytokeratins (CKs) by immunocytochemical staining on pancreatic tissue sections from normal rats, after partial pancreatectomy, and after isolation and culture of duct fragments. Monoclonal antibodies to human CK7, CK19, and CK20 were found to react specifically on rat pancreas tissue, as shown by Western blotting. CK20 and CK19 were immunocytochemically detected only in cells of the ductal system, from centroacinar cells to main ducts. CK7 was expressed by islets of Langerhans and by duct cells from main, inter-, and intralobular ducts, but not by centroacinar and terminal duct cells. CKs 7, 19, and 20 were also expressed in proliferating duct cells during tissue regeneration and after isolation and different periods of culture. We conclude that CKs 7, 19, and 20 are very useful markers to study the differentiation of rat duct cells under experimental conditions in vivo and in vitro.

Effect of dietary lipids and endocrine changes on polyunsaturated fatty acids in phospholipids of pancreas and brown adipose tissue of obese and lean rats

Obese (fa/fa) rats fed on control diet have lower proportions of linoleic acid (18:2n-6) and/or arachidonic acid (20:4n6) in IBAT and pancreas phospholipids compared with lean (Fa/-) rats. Lower stearic acid (18:0) to oleic acid (18:1n-9) mean ratios in fa/fa compared with Fa/- suggest enhanced Δ9-desaturase activity in the former. 18:2 20:4 mean ratios in pancreas, but not IBAT, are indicative of a reduced Δ6-desaturase activity in fa/fa rats. Absolute amounts of phospholipids (mg/unit tissue wt) were 2-fold greater in IBAT of fa/fa compared with Fa/- rats, irrespective of their diet. This was reflected in greater absolute amounts of 18:2n-6 and 20:4n-6 only in HSO and HTO fa/fa groups, but not in the control group. Adrenalectomy (Adx) or T 3 treatment also modified phospholipid fatty acid composition of IBAT and pancreas phospholipids in animals fed on the control diet with fa/fa rats more sensitive to endocrine induced changes. In fa/fa rats T 3 treatment increased docosahexaenoic acid (22:6n-3) in IBAT of both phenotypes compared with the control, but this effect was evident only in fa/fa and not Fa/- pancreas. T 3 treatment also increased docosapentaenoic acid (22;5n-3) in IBAT from, both phenotypes, but no 22:5n-3 was evident in the pancreatic tissue of these animals. ADX also increased 22:6n-3 in the IBAT of fa/fa (Fa/- values were less than 1%) but not in the pancreas of fa/fa or Fa/-. ADX modified the relative proportions of 18:0 to 18:1 and 18:2 to 20:4 in IBAT and pancreas of fa/fa in a way that indicated decreased Δ9-desaturase and increased Δ6-desaturase activities; these effects tended toward normal again in pancreas of fa/fa rats on corticosterone replacement (CST).

Characterization and clinical relevance of liver-pancreas antibodies in autoimmune hepatitis

The isolation of a marker antigen from rat liver and pancreas tissue, which reacts with antibodies in a subtype of autoimmune hepatitis by complement fixation test, enzyme-linked immunosorbent assay and Western blot, is described. The liver-pancreas antigen could be detected in tissue from different human or animal organs, but liver and pancreas yielded the highest activity. A highly specific antigen fraction was obtained by gel filtration and ion exchange chromatography with a 100,000 g supernatant from rat liver tissue, and this preparation was shown to be devoid of nuclear, mitochondrial and microsomal antigens and cytokeratin 8 and 18, as demonstrated by appropriate marker antibodies. These data and absorption studies with cell organelles indicate that liver-pancreas antigen is a cytosolic protein. By Western blotting, two major epitopes at molecular weights 52 kD and 48 kD could be visualized. Sera from 175 patients previously shown to have high complement-fixing activity to a nonpurified liver-pancreas antigen fraction were further analyzed. All were positive by enzyme-linked immunosorbent assay with the purified liver-pancreas antigen fractions, and 111 were also positive by Western blot. Eighty-six sera reacted with the 52-kD determinant, 33 with the 48-kD determinant and 2 with both determinants. In 117 of the 175 patients, antibody to liver-pancreas antigen was associated with other autoantibodies known to characterize subgroups of autoimmune hepatitis. Thus 19 patients had antibodies to nuclei and 96 to actin but none to liver-kidney microsomes, hereby suggesting that antibody to liver-pancreas antigen may define another subgroup of autoimmune hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of a monoclonal antibody recognizing the Golgi apparatus of serous exocrine cells.

To analyze in detail the dynamics of Golgi apparatus in exocrine cells, a monoclonal antibody against resident protein of the Golgi apparatus was prepared. A BALB/C mouse was immunized with fractions enriched in Golgi apparatus of guinea pig pancreas. The derived monoclonal antibody, GF-1, showed intense immunofluorescent staining of the Golgi area of rat pancreatic acinar cells by screening on cryosections. The antibody recognized a single polypeptide band on an immunoblot transferred from SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The polypeptide had a molecular weight of approximate ly 105-kDa and contained asparagine-linked carbohydrates. Light microscopic immunocytochemical study with rat pancreas, parotid gland, sublingual gland and gastric gland tissues indicated a staining pattern consistent with the Golgi apparatus but positive reaction was restricted to serous exocrine cells in these tissues. Immunoelectron microscopy with rat pancreatic and parotid acinar cells showed prominent staining of Golgi apparatus. The positive compartment of Golgi apparatus appeared to be its trans elements. It follows from these findings that monoclonal antibody GF-1 recognizes a glycopeptide of trans-Golgi membrane intimately related to specific functions of serous exocrine cells.

Staining of native and grafted exocrine rat pancreasby an interleukin-2 receptor specific monoclonal antibody

Immunohistological studies of native and grafted rat pancreas tissue with mouse anti-rat monoclonal antibodies revealed an unexpected staining pattern of MRC Ox 39 which binds specifically to rat interleukin-2 (IL-2) receptors. While the cytoplasm of acinar cells of native pancreas was stained, the lumina of the acini remained uncoloured. In grafted exocrine tissue, the staining pattern was quite reverse. Only the apical cytoplasm of acinar cells was stained while the acinar lumina exhibited an intense stain. Common binding sites shared between the IL-2 receptor and a hypothetical exportable protein in acinar cytoplasm could explain these observations.

Nicotinamide methylation tissue distribution, developmental and neoplastic changes

The distribution of cytosolic activity of nicotinamide: S-adenosylmethionine methyltransferase (nicotinamide methylase, EC 2.1.1.1) in normal tissues from adult rat and mouse and in tumors and the change in the enzyme activity during the the development of rat tissues were studied. (1) Rat liver exhibited the highest nicotinamide methylase activity among all adult tissues tested; other rat tissues, like adrenal, pancreas, kidney, brain and mouse tissues, had only less than 15% of the adult rat liver activity. (2) 3 days before birth, fetal liver showed a very low nicotinamide methylase activity (2% of adult rat liver), which, however, increased already 1 day before birth and reached the adult level on the day 28 after birth. (3) In a variety of hepatomas and ascites tumors, an inverse correlation, with some exceptions, between tumor growth rate and nicotinamide methylase activity could be seen. In all hepatomas, with the exception of Morris hepatoma 5123tc, nicotinamide methylase activity was significantly decreased in comparison to normal adult rat liver. The highly malignant Zajdela hepatoma, Yoshida sarcoma, sarcoma 180 and Ehrlich ascites tumor methylated nicotinamide only at a negligibly low rate. (4) Cultured RLC cells (an established rat liver cell line) from the stationary growth phase or G 1-arrested RLC cells had about half of the adult rat liver activity, yet the activity was 70% higher than that of the logarithmically growing RLC cells.

Hormonal stimulation of cAMP-dependent protein kinase in rat pancreas

Rat pancreas possesses a cAMP-dependent protein kinase enzyme which is inhibited by the protein kinase inhibitor from bovine heart. Incubation of rat pancreas tissue in the presence of secretin and CCK-PZ resulted in an increase of intracellular cAMP and activation of the cAMP-dependent protein kinase. CCK-OP, a pure synthetic analog of CCK-PZ, did not stimulate cellular cAMP production or protein kinase activation. There was correlation between the ability of a given dose of hormone to stimulate cAMP formation and activate the protein kinase enzyme. It is believed that the stimulatory effect of CCK-PZ is due to secretin contamination of this hormonal preparation.

Secretion in the B-cells of islets of Langerhans as demonstrated by zinc staining.

Intact islets of Langerhans were isolated from Wistar rats by means of vital fixation, microdissection and development of the metal sulfides formed with Timms reagent. In such intact islets, the beta-granules and the cell organelles can be shown up well electron microscopically. The endocrine pancreas tissue of normal rats contains a relatively constant average of 715 densely structured granules per 100 microns2. After administration of sulfonylurea, the number of (less dense) granules is reduced to 125 (in adipose animals 115) granules per 100 microns2. Besides empty 'sacs', large-vesicle Golgi apparatus and ballooned forms of the endoplasmic reticulum occur.

Changes in Somatostatin Concentration in Pancreas and Other Tissues of Streptozotocin Diabetic Rats*

Changes in immunoreactive somatostatin were examined in islets, whole pancreas, stomach, and hypothalamus of streptozotocin-diabetic rats. There was no change in islet somatostatin content at 2 days after the administration of streptozotocin, but thereafter, somatostatin progressively increased in the diabetic animals by 45% at 2 weeks, 230% at 6 weeks, and 500% by 6 months. By contrast, islet glucagon rose acutely and maintained a constant 2-fold elevation irrespective of the duration of the diabetes. Morphometric analysis of the somatostatin- and glucagon-producing cells in the islets revealed an apparent augmentation of both cell types. The concentration of somatostatin per total pancreas was also increased in the diabetic animals, suggesting that the islet increase was part of a true increase in pancreatic somatostatin. Pancreatic glucagon was unchanged despite the islet increase. The increase in pancreatic somatostatin was paralleled by an elevation in gastric somatostatin concentration, implying a common mechanism in response to streptozotocin for the somatostatin cells in these two sites. There was no change in hypothalamic somatostatin concentration. Islet somatostatin was also increased in alloxan-diabetic rats. suggesting that streptozotocin does not stimulate the D cells directly.

Effect of pilocarpine, carbamylcholine and pancreoxymin on the incorporation of 32P into phospholipids in rat pancreas tissue in vitro.

1. In the method described in the text, incorporation of 32P into phospholipids (PE, PC, Sph and Lyso-PC) in rat pancreas tissue was obserbed.2. In the case of pilocarpine, the incorporation of 32P into each phospholipid showed a tendency of increasing in comparison with the control.3. In the case of carbamylcholine, the incorporation of 32P into phospholipids generally increased, especially that into Lyso-PC remarkably in comparison with the control.4. In the case of pancreozymin, the incorporation of 32P into phospholipid showed generally a tendency of decreasing in comparison with the control except that into PC.The author is happy to acknowledge the advice given by Dr. Makoto Matsumoto, Dr. Sadahiko Ishibashi and Dr. Keizo Waku during these studies.He also thanks Miss Teruko Takahashi and Miss Yasue Odate for their able technical assistance.

In vitro synthesis of pancreatic enzymes: effect of soybean trypsin inhibitor.

ARTICLESIn vitro synthesis of pancreatic enzymes: effect of soybean trypsin inhibitorAM Konijn, Y Birk, and K GuggenheimAM Konijn, Y Birk, and K GuggenheimPublished Online:01 Apr 1970https://doi.org/10.1152/ajplegacy.1970.218.4.1113MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByThe Biochemical and Functional Food Properties of the Bowman-Birk Inhibitor14 February 2008 | Critical Reviews in Food Science and Nutrition, Vol. 48, No. 1Protease Inhibitors of Plant Origin and Role of Protease Inhibitors in Human Nutrition OverviewThe effect of fasting and cholecystokinin stimulation on normal and neoplastic tissue in the rat pancreasTissue and Cell, Vol. 24, No. 5Protein inhibitors of hydrolases in plant foodstuffs3 November 2009 | Food Reviews International, Vol. 6, No. 2Relevance of Multiple Soybean Trypsin Inhibitor Forms to Nutritional QualityPhotoreactive, Active Derivatives of Trypsin and Chymotrypsin Inhibitors from Soybeans and ChickpeasThe Bowman-Birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soybeans12 January 2009 | International Journal of Peptide and Protein Research, Vol. 25, No. 2Photoreactive derivative of Kunitz's soybean trypsin inhibitor.12 January 2009 | International Journal of Peptide and Protein Research, Vol. 23, No. 1Stimulation of Pancreatic Secretion and Growth in the Rat After Feeding CholestyramineGastroenterology, Vol. 83, No. 4SYNTHESIS, INTRACELLULAR TRANSPORT AND DISCHARGE OF EXPORTABLE PROTEINS IN THE PANCREATIC ACINAR CELL AND OTHER CELLSBiological Reviews, Vol. 53, No. 2Proteinase inhibitors of finger millet (Eleusine coracana gaertn.)Journal of the Science of Food and Agriculture, Vol. 29, No. 4Pancreatic Function and Disease in Infancy and ChildhoodAdvances in Pediatrics, Vol. 25, No. 1Oral Trypsin-inhibitor-induced Improvement of the Exocrine and Endocrine Pancreatic Functions in Alloxan Diabetic Rats9 February 2021 | Scandinavian Journal of Gastroenterology, Vol. 11, No. 4Regulation of pancreatic exocrine secretionPharmacological Research Communications, Vol. 8, No. 1[58] Trypsin and chymotrypsin inhibitors from soybeansNative and modified Bowman-Birk trypsin inhibitor—Comparative effect on pancreatic enzymes upon ingestion by quails (Coturnix coturnix japonica)Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, Vol. 48, No. 2Structure-Activity Relationships of Several Trypsin and Chymotrypsin Inhibitors from Legume SeedsThe neurohumoral control of pancreatic exocrine secretionThe American Journal of Clinical Nutrition, Vol. 26, No. 3A basic trypsin- and chymotrypsin-inhibitor from groundnuts (Arachis hypogaea)Biochimica et Biophysica Acta (BBA) - Protein Structure, Vol. 263, No. 3The interrelationships of the pancreatic enzymes.Gut, Vol. 13, No. 5Comparison of Pancreatic Changes Induced in Rats by Soybean and Egg-white Trypsin Inhibitors Fed with Different Levels of NitrogenThe Journal of Nutrition, Vol. 102, No. 4The effect of methionine supplementation on the levels of pancreatopeptidase E, trypsin, chymotrypsin and amylase in the pancreas of chicks receiving raw and heated soya-bean diets9 February 2010 | British Journal of Nutrition, Vol. 27, No. 2Effect of caerulein on protein synthesis and secretion in the guinea-pig pancreas19 July 2012 | British Journal of Pharmacology, Vol. 40, No. 4 More from this issue > Volume 218Issue 4April 1970Pages 1113-1117 Copyright & PermissionsCopyright © 1970 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1970.218.4.1113PubMed5461613History Published online 1 April 1970 Published in print 1 April 1970 Metrics

Fate of ascorbic acid in early radiation damage.

1. There were no gross changes in concentration or total content of ascorbic acid within 72 hours after 600 r of X-ray in rat liver, brain, pancreas, testis, muscle, kidney, or brown adipose tissue. Thymus, lymph nodes, and spleen showed a drop in ascorbic acid concentration within four hours, and the content of ascorbic acid continued to drop throughout the period studied. Intestinal mucosa showed a sharp increase in ascorbic acid concentration during the first 4 hours followed by a gradual decline. 2. Stimulation of ascorbic acid synthesis by chloretone administration does not modify the effects of irradiation, nor does the hydration of these tissues change in a fashion which might account for the observed phenomena. 3. The observed changes in concentration could not be duplicated by the administration of aminopterin at a level of 10 mg/kg body weight.

The cultivation in fluid medium of organised liver, pancreas and other tissues of foetal rats

Summary A modification of the classical watch-glass method for tissue culture is described, in which the explants are grown on lens paper floating on a liquid medium. During subcultivation, the lens paper with its adherent explants is lifted bodily from the liquid, washed in Tyrode and laid on fresh culture medium. The method is especially suitable for growing highly secretory tissues in vitro , as it ensures easy disposal of waste products from the explants and minimises disturbance to the tissue during subcultivation. Fragments of liver and pancreas from foetal rats grew and differentiated extremely well in vitro . Good results have also been obtained with primordia of the thyroid, anterior pituitary gland, the adrenal cortex, spleen and lung.