Rolf Eissele

Glucagon-like peptide-1 cells in the gastrointestinal tract and pancreas of rat, pig and man

Abstract. A highly specific monoclonal antibody directed against the C‐terminal part of glucagon‐like peptide‐1 (GLP‐1) was raised to immunohistochemi‐cally evaluate the distribution of GLP‐1 containing cells in the entire gastrointestinal tract including pancreas of rat, pig and man. In the pancreas GLP‐1 ‐immunoreactive cells were found variously shaped and predominantly located in the periphery of the islets. Ultrastructurally, GLP‐1 was co‐localized with gluca‐gon in the α‐granula of A‐cells and was mainly restricted to the electrondense core. In the intestine open type cells reaching the lumen via a slender apical process were stained with the GLP‐1 antibody. They occurred in all parts of the crypts but predominantly in the basal portion. The density of GLP‐1 immuno‐reactive cells varied between species in a characteristic order: rat > pig > man. In pig and human gut a large number of cells occurred in the distal jejunum and ileum. A continuous increase of cell densities was found from the proximal to the distal colon resulting in highest numbers in the rectum. In rats the highest cell density occurred in the ileum. Again, a continuous increase of GLP‐1‐positive cell numbers was evident from the proximal to the distal portion of small and large bowel. GLP‐1 was partly co‐localized with PYY. The GLP‐1 positive cells appeared electronmicrosco‐pically as L‐cells with the typical large granula. This morphological data indicates that GLP‐1‐releasing cells in the small intestine are appropriately positioned in the distal part to sense and respond to the presence of nutrients that have escaped the absorptive surface of the upper small intestine.

Expression of the two isoforms of the vesicular monoamine transporter (VMAT1 and VMAT2) in the endocrine pancreas and pancreatic endocrine tumors

The uptake of monoamines into the secretory granules of monoamine-storing neuroendocrine cells is mediated by vesicular monoamine transporter protein 1 or 2 (VMAT1 or VMAT2). This study analyzed the expression of VMAT1 and VMAT2 in endocrine cells of normal human and monkey pancreas. The expression of VMAT1 and VMAT2 was also examined in infants with hyperinsulinemic hypoglycemia and in adults with pancreatic endocrine tumors (PETs). Using immunohistochemistry (IHC) and in situ hybridization (ISH), we demonstrated the mutually exclusive expression of VMAT1 in endocrine cells of the duct system and of VMAT2 in many cells of the islets of Langerhans. By confocal laser scanning microscopy, VMAT1-positive cells were identified as enterochromaffin (EC) cells and VMAT2-positive cells as β-cells. In PETs, VMAT1 was found exclusively in all serotonin-containing tumors. In contrast, VMAT2 expression was lost in many insulinomas, independent of their biological behavior. VMAT2 was expressed by some non-insulin-producing tumors. The mutually exclusive expression of VMAT1 in EC cells and of VMAT2 in β-cells suggests that both cell types store monoamines. Monoamine storage mediated by VMAT1 in EC cells is apparently maintained in EC cell tumors. In contrast, many insulinomas appear to lose their ability to accumulate monoamines via VMAT2.

Rat gastric somatostatin and gastrin release: Interactions of exendin-4 and truncated glucagon-like peptide-1 (GLP-1) amide

The effect of exendin-4, a peptide of the secretin-glucagon family with high homology of amino acid sequence with glucagon-like peptide-1 (GLP-1), on gastric hormone release was investigated in the isolated perfused rat stomach. Exendin-4 dose dependently stimulated somatostatin release up to 9-fold at a concentration of 10(-7) M whereas gastrin release was inversely inhibited by up to 63%. These effects could partially be reduced by concomitant perfusion of truncated exendin-4, exendin(9-39)amide. Similarly, stimulation of somatostatin secretion and inhibition of gastrin release induced by GLP-1(7-36)amide was partially reversed by exendin-4 (9-39)amide. These data are consistent with the assumption that exendin-4 and truncated GLP-1amide exert their effects on gastric D and G cell by interaction with the same receptor.

Islet amyloid polypeptide (IAPP ; amylin) influences the endocrine but not the exocrine rat pancreas

The effect of synthetic rat amylin (10,100,1000 pmol/l) on glucose (10 mmol/) and arginine (10 mmol/l) -stimulated islet hormone release from the isolated perfused rat pancreas and on amylase release from isolated pancreatic acini was investigated. Amylin stimulated the insulin release during the first (+76%) and the second secretion period (+42%) at 1 nmol/l. The first phase of the glucagon release was inhibited concentration dependently by amylin and completely suppressed during the second phase. Amylin diminished the somatostatin release in a concentration dependent manner. This effect was more pronounced at the first than the second secretion period (1 nmol amylin: 1 phase: -60%, 2.phase: -22%). Amylin was without any effect on basal and CCK stimulated amylase release from isolated rat pancreatic acini. Our data suggest amylin, a secretory product of pancreatic B-cells, as a peptide with approximately strong paracrine effects within the Langerhans islet. Therefore, amylin might be involved in the regulation of glucose homeostasis.

Expression and cell-specific localization of the cholecystokinin B/gastrin receptor in the human stomach.

Abstract. Gastrin stimulates gastric acid secretion by acting on the cholecystokinin B/gastrin receptor (CCK-BR). The localization of this receptor at the cellular level showed conflicting results in animal studies and has not been described in man by immunohistochemistry. The aim of the present study is to characterize the precise cellular location of the CCK-BR in the human stomach. Polyclonal antisera were raised against different epitopes of the CCK-BR molecule and used for immunohistochemical investigations. CCK-BR mRNA was detected in paraffin tissue sections by the highly sensitive method of in situ reverse transcriptase-polymerase chain reaction (RT-PCR). Using immunohistochemistry, CCK-BR could successfully be localized in gastric parietal cells. In the majority of parietal cells, CCK-BR immunoreactivity was present at the basolateral cell membrane domain. In some parietal cells, a granular pattern of immunoreactivity was exclusively confined to the cytoplasm of the cells. CCK-BR mRNA was found in parietal cells and in enterochromaffin-like (ECL) cells by means of in situ RT-PCR. No expression of CCK-BR was found in the gastric antral mucosa. Our data support the concept that gastrin stimulates gastric acid secretion directly via CCK-B receptors on parietal cells and indirectly by inducing histamine release from histamine-containing ECL cells, which contributes to acid secretion by parietal cells.

Expression of Vesicular Monoamine Transporters in Endocrine Hyperplasia and Endocrine Tumors of the Oxyntic Stomach

Background: Gastric enterochromaffin-like (ECL) cells selectively express the vesicular monoamine transporter (VMAT) VMAT2, and enterochromaffin (EC) cells the VMAT1 isoform. Aims: We investigated whether VMAT isoform selection indicates the origin of endocrine hyperplasia and neoplasia from oxyntic ECL or EC cells and may be of prognostic significance in different types of gastric carcinoids. Methods: Tissue from patients with chronic atrophic gastritis (CAG), Zollinger-Ellison-syndrome (ZES), gastric carcinoids and neuroendocrine carcinoma (NEC) was investigated by immunohistology and in situ hybridization. Results: Endocrine cells forming diffuse, linear, and micronodular hyperplasia in CAG and ZES, as well as oxyntic microcarcinoids expressed both VMAT2 and chromogranin A (CgA) but neither VMAT1 nor serotonin. In five of six sporadic carcinoids VMAT2 and CgA but not VMAT1 were detected. One carcinoid was copositive for VMAT1 and serotonin but negative for VMAT2. Electron microscopy confirmed the VMAT2-positive tumors as ECLoma and the VMAT1-immunoreactive carcinoid as EComa. Conclusions: VMAT2 and VMAT1 are reliable markers for differentiation of gastric endocrine hyperplasia and neoplasia from ECL and EC cells, respectively. The significance of VMAT2 and VMAT1 as prognostic markers lies in the relatively poor prognosis for EComa compared to ECLoma, characterized by VMAT2 positivity. The absence of both VMAT2 and VMAT1 in NEC may indicate poor prognosis.

Influence of chronic drug-induced achlorhydria by substituted benzimidazoles on the endocrine stomach in rats

The release of gastric somatostatinlike immunoreactivity and gastrin was studied in rats with chronic achlorhydria induced by the substituted benzimidazole BY 308. In vitro, stimulation of gastrin release by acetylcholine was slightly enhanced after 1 day of treatment but no further effects were observed compared to placebo controls. Four weeks of treatment evoked marked gastrin hypersecretion, which was atropine-resistant. Stimulation of gastrin release was inversely correlated to enhancement of basal gastrin levels. Chronic achlorhydria distinctly reduced somatostatin responses to isoproterenol, whereas potent stimulation was observed in controls. Treatment with BY 308 for 1 wk was associated with fully developed gastrin hypersecretion but isoproterenol-stimulated somatostatin release was still unaffected. Hypergastrinemia accompanied by increased antral gastrin and reduced antral and fundic somatostatin concentrations was also found in vivo after 4 wk of treatment with BY 308. It is concluded that chronic achlorhydria not only enhances storage and secretion of gastrin but also diminishes the secretion and tissue stores of somatostatin; adaptive changes of the somatostatin cell occur, however, with a much longer delay.

Proliferation of endocrine cells in the rat stomach caused by drug-induced achlorhydria

Time-related changes of serum gastrin levels, gastrin cell, and enterochromaffinlike cell densities, and proliferation kinetics of these cells have been examined in rats during treatment with the substituted benzimidazole BY 308 over a period of 73 days. Serum gastrin levels increased very rapidly from 74 +/- 6 pg/mL (controls) to 438 +/- 31 pg/mL (day 1) and 727 +/- 68 pg/mL (day 4). Thereafter, a steady increase was observed until day 70 (2097 +/- 208 pg/mL). Enterochromaffinlike cell density was unchanged until day 10, but then increased progressively without reaching a plateau (144% above control on day 73). The labeling index of these cells was enhanced shortly after drug application and remained on a constant elevated level from day 14 to day 73 (about 10-fold to 12-fold above controls from day 14 to day 70). The number of gastrin cells increased rapidly within the first week and reached a plateau after 17 days (96% increase above controls). In contrast to enterochromaffinlike cells, the labeling index did not change immediately but increased on day 7 by 37% and returned beneath control values after day 28. The results suggest that in drug-induced achlorhydria, the progressive increase of enterochromaffinlike cells is explained by an enhanced mitosis, whereas other factors in addition to proliferation are responsible for the augmentation of gastrin cells.

Reciprocal cellular distribution of glucagon-like peptide-1 (GLP-1) immunoreactivity and GLP-1 receptor mRNA in pancreatic islets of rat.

The respective cellular distribution of glucagon-like peptide-1 (GLP-1) immunoreactivity and mRNA expression of the GLP-1 receptor was compared in rat pancreas by means of immunohistochemistry and in situ hybridization. GLP-1 immunoreactivity was present in the marginal zone of rat pancreatic islets. In contrast, GLP-1 receptor mRNA signals were confined to the central part of pancreatic islets. Neither GLP-1 immunoreactivity nor GLP-1 receptor mRNA signals were detected in the exocrine pancreatic acinar cells or duct cells. The differential distribution of GLP-1 immunoreactivity and GLP-1 receptor mRNA signals indicates that the GLP-1 amino acid sequence is present in the α-cell zone of pancreatic islets, whereas the GLP-1 receptor is expressed mainly by α cells. Thus, our data by in situ hybridization demonstrates the significant expression of GLP-1 receptors on β cells but makes a significant expression on α cells rather unlikely.

Calcitonin gene-related peptide stimulates rat gastric somatostatin release in vitro

The influence of rat calcitonin gene-related peptide (rCGRP) on the secretion of gastric somatostatin and gastrin was studied in vitro using the isolated, vascularly perfused rat stomach preparation. rCGRP stimulated somatostatin secretion dose-dependently reaching 3-fold stimulation at 1 microM. The kinetics of somatostatin response were characterized by a sharp increase in the initial phase of rCGRP perfusion followed by sustained elevated levels. Gastrin secretion was moderately suppressed at 1 nM to 100 nM CGRP. Somatostatin responses to half-maximal stimulation with 100 nM CGRP were not affected by concomitant perfusion of atropine, propranolol, and tetrodotoxin. It is concluded that increases in somatostatin release in response to CGRP are probably due to a direct effect on the gastric somatostatin-producing D-cell and may be important for the potent acid-inhibitory activity of CGRP.