Immunohistochemical localization of gamma-aminobutyric acid (GABA) in the rat pancreas

The localization of gamma-aminobutyric acid (GABA) in rat pancreas was investigated using antiserum raised against GABA conjugated to bovine serum albumin with glutaraldehyde. Immunoreactive cells were only found in the center of the pancreatic islets, and these cells were surrounded by nonimmunoreactive cells. When two serial sections of rat pancreas were consecutively stained with GABA antiserum and with antibodies against insulin, both antisera stained the same population of endocrine cells within the islets. In rats pretreated with streptozotocin, a B-cell toxin, we observed a marked decrease in the number of cells exhibiting GABA-like immunoreactivity. These observations indicate that GABA is present in the B cells of rat pancreatic islets.     

GABA in the endocrine pancreas: cellular localization and function in normal and diabetic rats

Gamma amino butyric acid (GABA) and its related enzymes have been demonstrated in pancreatic beta cells of normal rat. Antibodies against GABA-synthesizing enzymes have been implicated in the pathogenesis of Type I diabetes. In spite of the importance of GABA in the aetiology of diabetes mellitus, detailed morphological data on the pattern of distribution of GABA in the pancreas of normal and diabetic rats are lacking. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (60 mg kg body weight(-1)). Four weeks after the induction of DM, normal (n = 6) and diabetic (n = 6) rats were anesthetized with chloral hydrate and their pancreata were removed and processed for the localization and effect of GABA on insulin secretion using immunohistochemistry and radioimmunoassay techniques. The number of GABA-like immunoreactive (GABA-LIR) cells in the pancreatic islets of STZ-diabetic rats decreased significantly (P<0.0001) when compared to non-diabetic control rats. The pattern and percentage distribution of GABA in the islet of Langerhans of normal and diabetic rat was similar to that of insulin. GABA induced a significant (P<0.0007) increase in insulin secretion from the pancreas of normal rats. In diabetic pancreas, GABA evoked a higher but not significant (P<0.1) increase in insulin secretion. These findings showed that the number of GABA-LIR cells is reduced significantly in diabetes. Moreover, GABA is a strong secretagogue of insulin from the pancreas of normal rat.     

An immunocytochemical study of endocrine pancreas of snakes

Summary The pancreas from eleven species of snakes representing both advanced and primitive families has been investigated for the presence of eleven regulatory peptides reported to occur in the mammalian endocrine pancreas. Of the eleven peptides studied, insulin, pancreatic glucagon and somatostatin were present in endocrine cells within the islets of all the species investigated. The neuropeptide, vasoactive intestinal polypeptide, was located within nerve terminals innervating the islets in the Boidinae, Colubrinae, Elaphidae and Crotalidae but absent from the Natricinae investigated.No immunoreactivity was demonstrable with the antisera to substance P, met-enkephalin, C-terminal gastrin, bombesin, glicentin and gastric inhibitory polypeptide. Pancreatic polypeptide-like immunoreactivity was demonstrable only in the boid snakes and exclusively stained by a C-terminal specific antiserum.     

Effects of gamma-aminobutyric acid on secretagogue-induced exocrine secretion of isolated, perfused rat pancreas

Because GABA and its related enzymes have been determined in beta-cells of pancreas islets, effects of GABA on pancreatic exocrine secretion were investigated in the isolated, perfused rat pancreas. GABA, given intra-arterially at concentrations of 3, 10, 30, and 100 microM, did not exert any influence on spontaneous or secretin (12 pM)-induced pancreatic exocrine secretion. However, GABA further elevated CCK (10 pM)-, gastrin-releasing peptide (100 pM)-, or electrical field stimulation-induced pancreatic secretions of fluid and amylase dose dependently. The GABA (30 microM)-enhanced CCK-induced pancreatic secretions were completely blocked by bicuculline (10 microM), a GABA(A) receptor antagonist, but were not affected by saclofen (10 microM), a GABA(B) receptor antagonist. The enhancing effects of GABA (30 microM) on CCK-induced pancreatic secretions were not changed by tetrodotoxin (1 microM) but were partially reduced by cyclo-(7-aminoheptanonyl-Phe-D-Trp-Lys-Thr[BZL]) (10 nM), a somatostatin antagonist. In conclusion, GABA enhances pancreatic exocrine secretion induced by secretagogues, which predominantly induce enzyme secretion, via GABA(A) receptors in the rat pancreas. The enhancing effect of GABA is partially mediated by inhibition of islet somatostatin release.     

VGF metabolic-related gene: distribution of its derived peptides in mammalian pancreatic islets.

The vgf gene has been shown to be involved in several metabolic pathways. Because the pancreas is crucial to metabolism and food intake, we studied the VGF peptides in bovine, rat, and pig Langherans islets using antisera raised against specific sites along the primary sequence of the rat/mouse and human VGF protein precursor. Whereas almost all of the pancreatic endocrine cells expressed vgf mRNA, when using the VGF antisera a different staining pattern became apparent. VGF(556-565) and VGF(282-291) immunoreactivity were exclusively found in delta somatostatin-producing cells, whereas the human C-terminus antiserum selectively immunolabeled alpha glucagon and pancreatic polypeptide cells. The same cells were decorated with the VGF(443-588) antiserum, which also weakly labeled beta insulin-secreting cells. Finally, the VGF(298-306) peptide and the rat C terminus were found in virtually all pancreatic endocrine cells. Using bovine, swine, and rat pancreatic extracts, data from chromatography and ELISA assay showed the presence of a high molecular mass form compatible with the proVGF and lower molecular mass fractions corresponding to short VGF peptides. In conclusion, selective VGF distribution may suggest a multifaceted cell type-specific processing of proVGF, resulting in different peptides probably involved in neuroendocrine regulatory metabolic mechanisms.     

Autoradiographic studies of the gamma-aminobutyric acid (GABA) system in the rat pancreas

The beta-cells of the pancreatic islets have been shown to contain gamma-aminobutyric acid (GABA) together with insulin. Autoradiographic analysis indicated that high affinity GABA binding sites (GABA receptors) are not present in the pancreas. High affinity GABA uptake sites are present, not in beta-cells, but in a few cells on the periphery of the islets. These observations cast doubt on the suggestion that GABA has a paracrine role in the pancreas.     

Oxytocin-like immunoreactive nerves are associated with insulin-containing cells in pancreatic islets of anglerfish (Lophius americanus)

Summary Recent reports indicate that oxytocin exerts direct effects on the release of insulin and glucagon from the endocrine pancreas of the rat. The purpose of this study was to determine whether oxytocin-like immunoreactivity is present in the anglerfish islet, and if it is associated with subsets of hormone-producing cells. Antisera against oxytocin, insulin, glucagon, somatostatin, neuropeptide Y, and the 200 — kd neurofilament polypeptide were applied to serial 5 m sections of pancreatic islets. The antiserum to the 200 — kd neurofilament polypeptide labeled nerve bundles and axons, some of which were also stained with the oxytocin antiserum. Oxytocin immunoreactivity was observed in large nerves that branched into varicose fibers. These fibers were consistently associated only with clusters of insulin-producing cells. Successive application of oxytocin and insulin antisera to the same section provided additional verification of this relationship. Oxytocin-labeled nerves were not associated with cells immunoreactive to glucagon, somatostatin, or neuropeptide Y (anglerfish peptide Yg). The results demonstrate that oxytocin or an oxytocin-like peptide is located in fibers that surround only insulin-producing cells in the anglerfish islet. Although the functional significance of this observation remains to be determined, the results imply that oxytocin, or an oxytocin-like peptide, may affect the synthesis or release of insulin from anglerfish islets.     

Pancreatic polypeptide-like material in nerves and endocrine cells of the rat

A region-specific antiserum (AbS11) raised against the carboxyl-terminal hexapeptide of pancreatic polypeptide has been employed to measure rat pancreatic polypeptide specifically and to demonstrate apparent immunoreactivity in nerves and in endocrine cells outside the pancreas. The concentration of pancreatic polypeptide in the head of the rat pancreas measured with AbS11 (176 +/- 47 pmol/g) was 750 fold higher than that measured with a conventional anti-bPP antiserum (0.23 +/- 0.08 pmol/g). Column chromatographs of rat pancreatic extracts demonstrated two peaks of immunoreactivity both eluting after the porcine pancreatic polypeptide standard. AbS11 also detected specific immunoreactivity in rat brain (470 fmol/g) which went undetected in convention assays. Although immunohistochemical studies with AbS11 and human pancreatic polypeptide antiserum demonstrated immunoreactivity in the same population of pancreatic endocrine cells, immunoreactive nerve fibres and enteroglucagon cells were only demonstrable with AbS11. These studies demonstrate that the carboxyl terminus of rat pancreatic polypeptide is immunochemically similar to that of higher mammals. Furthermore, neural and extrapancreatic endocrine variants of this peptide share an immunochemical determinant contained within the carboxyl-terminal hexapeptide.     

Expression and distribution of somatostatin receptor subtypes in the pancreatic islets of mice and rats.

Somatostatin acts on specific membrane receptors (sst(1-5)) to inhibit exocrine and endocrine functions. The aim was to investigate the distribution of sst(1-5) in pancreatic islet cells in normal mice and rats. Pancreatic samples from five adult C57BL/6 mice and Sprague-Dawley rats were stained with antibodies against sst(1-5) and insulin, glucagon, somatostatin, or pancreatic polypeptide (PP). A quantitative analysis of the co-localization was performed. All ssts were expressed in the pancreatic islets and co-localized on islet cells to various extents. A majority of the beta-cells expressed sst(1-2) and sst(5) in mouse islets, while < or =50% in the rat expressed sst(1-5). The expression of sst(1-5) on alpha-cells did not differ much among species, with sst(2) and sst(5) being highly expressed. About 70% of the delta-cells expressed sst(1-4) in the rat pancreas, whereas 50% of the islet cells expressed sst(1-5) in the mouse. Furthermore, 60% of the PP-cells expressed sst(1-5) in the mouse, while the rat islets had lower values. Co-expression with the four major islet hormones varies among species and sst subtypes. These similarities and differences are interesting and need further evaluation to elucidate their physiological role in islets.     

大鼠胰腺嗜铬颗粒素A分布的免疫组织化学研究

本研究用ＡＢＣ免疫组织化学方法,在Ｂｏｕｉｎ液固定的常规石蜡切片上,观察了啥铬颗粒素Ａ在大鼠胰腺内分泌细胞内的定位和分布,并用相邻切片双标记法,观察了它与胰高血糖素、胰岛素、生长抑素的共存关系。结果发现,大鼠胰腺嗜铬颗粒素Ａ样免疫反应细胞主要分布于胰岛的周边部,胰腺外分泌部的导管和腺泡等处均未见ＣｇＡ祥物质存在。用相邻薄切片免疫显色技术证明,大鼠胰腺中ＣｇＡ样物质与胰高血糖素共存。结果提示,ＣｇＡ可能是胰腺内分泌细胞的一个新的标志物,在胰腺功能调节上发挥着重要作用。     

Immunoreactive GABA transaminase within the pancreatic islet is localized in mitochondria of the B-cell

Subcellular localization of gamma aminobutyrate-alpha-ketoglutarate transaminase (GABA-T) in the pancreatic islets of Langerhans was determined by use of an electron microscopic, immunogold post-embedding protocol. The objective of this study was to define the islet cell distribution and subcellular localization of GABA-T. Within the islet, GABA-T was found only in the B-cells and was localized in mitochondria; 78 mitochondria contained 336 gold particles, whereas 245 secretory granules contained only 18 gold particles. Although studies utilizing either the isolated perfused pancreas or cultured islets have shown that exogenous GABA modulates D-cell secretion, in this study immunoreactive GABA-T, the catabolic enzyme for GABA, was not detectable in A- and D-cells of the islet. Control studies substituting normal rabbit serum for the GABA-T antiserum resulted in absence of labeling. These results indicate that the high concentration of GABA present in islet B-cells is catabolized by GABA-T in the mitochondrial compartment, consistent with the possibility that GABA functions as a mediator of B-cell activity.     

Zinc-Specific N-(6-Methoxy-8-Quinolyl)-Para-Toluenesulfonamide as a Selective Nontoxic Fluorescence Stain for Pancreatic Islets

Separation of the endocrine from the exocrine pancreatic tissue by fluorescence activated sorting has been limited by the lack of an ideal fluorescent label for islet tissue. Our studies indicates the zinc-specific stain N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), has characteristics ideal for use as a fluorescent label for islet tissue. Dispersed rat pancreas cells stained with TSQ produced bright blue fluorescence when excited by UV light [peak emission wavelength at 480 nm. maximal excitation at 365 nm). The fluorescence was specific for islet tissue as confirmed by counterstaining with the islet-specific stain dithizone and there was minimal background staining of exocrine tissue. Stained tissue remained brightly fluorescent for 2 hr. with some fading by 4 hr. Injection of TSQ into rats at a concentration sufficient to produce staining of islets produced no toxicity discernible at 4 months. The viability of isolated rat islets stained with TSQ was maintained as shown by supravital staining, in vitro secretion of insulin, and reversal of diabetes after transplantation of stained islets into diabetic syngeneic recipients.     

Stains for A, B, and D cells in fetal rat islets.

Ten techniques often used for identification of A, B, and D cells in adult islets of Langerhans were applied to fetal rat pancreas. Modifications were tried with many of these techniques. Two indole methods (xanthydrol and postocoupled benxylidene reactions) and a cryostat technique using o-phthaladehyde failed to stain fetal islets. Phosphotungstic acid hematoxylin and lead hematoxylin lightly stained fetal A cell granules in Helly's fixed tissue. The Grimelius silver nitrate technique stains adult rat A cells but failed to stain fetal cells. A modification of this technique stained fetal A cells and a possible 4th cell type. The specificity of this method was confirmed by restaining stained cells with a fluorescent antibody technique and with pseudoisocyanin. B cells, as previously reported, were readily stained by the aldehyde fuchsin technique. Fetal D cells were not stained by the Hellerstrom-Hellman alcoholic silver nitrate method, nor did they display pseudoisocyanin metachromasia after acid hydrolysis; they did fluoresce brightly with this technique when viewed with UV light. It was thus possible to distinguish the three usual cell types, plus a possible fourth type, in the fetal rat pancreas.     

Ontogeny of immunoreactive glicentin in the human gastrointestinal tract and endocrine pancreas

The gestational time of appearance and distribution of immunoreactive glicentin was compared to that of immunoreactive glucagon in the gastrointestinal tract and endocrine pancreas of human fetuses, aged between 5 and 24 weeks, by an indirect immunoperoxidase method. With the glicentin antiserum No. R 64, the first immunoreactive cells were detected at the 10th week of gestation in the oxyntic mucosa and proximal small intestine, at the 8th week in the ileum and at the 12th week in the colon. In the endocrine pancreas, the first immunoreactive cells were observed as early as 8 weeks within the walls of the primitive pancreatic ductules. At a more advanced stage of development (12 weeks), they were found interspersed among the islet cell clusters and still later (16 weeks) inside the recognizable islets of Langerhans. With the glucagon antiserum No. GB 5667, no immunoreactive cells were demonstrated in the gastrointestinal tract whatever the age of the fetuses. In the endocrine pancreas, the first immunoreactive cells were observed at the 8th week of gestation in the pancreatic parenchyma. The distribution of glucagon-containing cells in the pancreas was similar to that of glicentin immunoreactivity throughout ontogenesis. In the pancreatic islets of one 18-week-old human fetus, the study of consecutive semithin sections treated by both antisera showed that the same cells were labelled. The significance of these findings concerning the role of glicentin as a glucagon precursor is discussed.     

A lysophospholipase specific for exocrine pancreatic cells is stored in zymogen granules and secreted into pancreatic juice

We separated by two-dimensional (2D) gel electrophoresis the content of isolated rat zymogen granules and from the gel excised a protein of apparent MW 77,500 and an isoelectric point of about 4.7. A rabbit antiserum against this previously uncharacterized rat zymogen granule protein recognized two cDNA clones in a rat pancreas expression library. The cDNA inserts of these two clones had sequences showing perfect homology to the published cDNA sequence of rat pancreatic lysophospholipase. The antiserum recognized only a single protein, lysophospholipase, on one and two-dimensional immunoblots of rat pancreas homogenates and isolated zymogen granules. The antiserum did not react with any protein in homogenates of rat liver, spleen, adrenal, parotid, and prostate tissue. The zymogen granule protein of the guinea pig, previously identified as Lipase 1, was recognized specifically by the antiserum against rat lysophospholipase. This guinea pig protein can now be regarded as lysophospholipase. The same protein was demonstrated in the transformed rat acinar cell line AR4-2J, where both the rate of total enzyme synthesized and the amount of mRNA increased following treatment with dexamethasone. Immunogold labeling established that pancreatic lysophospholipase is restricted exclusively to exocrine cells where it occurs only in compartments of the exocytotic pathway. It could also be detected in pancreatic juice in the ducts of the tissue. Finally, we have shown that lysophospholipase is not related to the zymogen granule membrane protein GP2. This work establishes that lysophospholipase is a normal member of the set of soluble enzymes and proenzymes that are stored in zymogen granules and secreted into pancreatic juice.     

Anglerfish islets contain NPY immunoreactive nerves and produce the NPY analog aPY

It has recently been demonstrated that aPY, a peptide which has significant homology with neuropeptide Y (NPY) is present in extracts of anglerfish islets. The purpose of this study was to determine whether cells or nerves which contain NPY-like immunoreactivity could be identified in anglerfish islet tissue and whether aPY is synthesized by this tissue. Antisera against bovine pancreatic polypeptide (BPP), NPY and the 200 kd neurofilament polypeptide were used for immunohistochemical analysis of islets. Identical cells were stained by both the NPY and BPP antisera. The NPY and 200 kd neurofilament antisera also labeled nerve fibers in the tissue which were not stained with the BPP antiserum. The nature of the NPY-like peptide synthesized in islet cells was determined by subjecting differentially radioactively labeled Mr 2,500-8,000 peptides from islet extracts to reverse phase HPLC. Labeled aPY was unequivocally identified in the extracts and was labeled appropriately (as predicted from its sequence) with 13 different radioactive amino acids. These results demonstrate that one form of NPY-like peptide synthesized in anglerfish islets is aPY. The form of NPY-like peptide which was immunolocalized in nerves remains to be determined.     

In vitro and in vivo protective effect of Ganoderma lucidum polysaccharides on alloxan-induced pancreatic islets damage

This study was undertaken to investigate the protective effect against alloxan-induced pancreatic islets damage by Ganoderma lucidum Polysaccharides (Gl-PS) isolated from the fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst. In vitro, alloxan caused dose-dependent toxicity on the isolated pancreatic islets. Pre-treatment of islets with Gl-PS for 12 h and 24 h significantly reversed alloxan-induced islets viability loss. Gl-PS was also found to inhibit the free radicals production induced by alloxan in the isolated pancreatic islets using confocal microscopy. Gl-PS dose-dependently increased serum insulin and reduced serum glucose levels when pretreated intragastrically for 10 days in alloxan-induced diabetic mice. It was found that the pancreas homogenates had higher lipid peroxidation products in alloxan-treated mice than in the Gl-PS-treated animals. Aldehyde fuchsin staining revealed that alloxan caused nearly all the beta cells disappearing from the pancreatic islets, while Gl-PS partly protected the beta cells from necrosis. Alloxan (60 mg/kg) induced NF-kappa B activation in the pancreas at 30 min after injection, pretreatment with Gl-PS inhibited alloxan-induced activation of NF-kappa B. These results suggest that Gl-PS was useful in protecting against alloxan-induced pancreatic islets damage in vitro and in vivo; one of the mechanisms is through its scavenging ability to protect the pancreatic islets from free radicals-damage induced by alloxan.     

Selective Expression of Huntingtin-associated Protein 1 in ��-Cells of the Rat Pancreatic Islets

Huntingtin-associated protein-1 (HAP1) was initially identified as a binding partner of huntingtin, the Huntington''s disease protein. Based on its preferred distribution among neurons and endocrine cells, HAP1 has been suggested to play roles in vesicular transportation in neurons and hormonal secretion of endocrine cells. Given that HAP1 is selectively expressed in the islets of rat pancreas, in this study, we analyzed the expression pattern of HAP1 in the islets. In rats injected intraperitoneally with streptozotocin, which can selectively destroy β-cells of the pancreatic islets, the number of HAP1 immunoreactive cells was dramatically decreased and was accompanied by a parallel decrease in the number of insulin-immunoreactive cells. Immunofluorescent double staining of pancreas sections showed that, in rat islets, HAP1 is selectively expressed in the insulin-immunoreactive β-cells but not in the glucagon-immunoreactive α-cells and somatostatin immunoreactive δ-cells. In isolated rat pancreatic islets, ∼80% of cells expressed both HAP1 and insulin. Expression of HAP1 in the INS-1 rat insulinoma cell line was also demonstrated by immunofluorescent staining. Western blotting further revealed that HAP1 in both the isolated rat pancreatic islets and the INS-1 cells also has two isoforms, HAP1A and HAP1B, which are the same as those in the hypothalamus. These results demonstrated that HAP1 is selectively expressed in β-cells of rat pancreatic islets, suggesting the involvement of HAP1 in the regulation of cellular trafficking and secretion of insulin. (J Histochem Cytochem 58:255–263, 2010)