Gastroenteropancreatic Endocrine Cells Research Articles

VEGF Secretion by Neuroendocrine Tumor Cells Is Inhibited by Octreotide and by Inhibitors of the PI3K/AKT/mTOR Pathway

Gastroenteropancreatic (GEP) endocrine tumors are hypervascular tumors able to synthesize and secrete high amounts of VEGF. We aimed to study the regulation of VEGF production in GEP endocrine tumors and to test whether some of the drugs currently used in their treatment, such as so- matostatin analogues and mTOR inhibitors, may interfere with VEGF secretion. We therefore analyzed the effects of the somatostatin analogue octreotide, the mTOR inhibitor rapamycin, the PI3K inhibitor LY294002, the MEK1 inhibitor PD98059 and the p38 inhibitor SB203850 on VEGF secretion, assessed by ELISA and Western blotting, in three murine endocrine cell lines, STC-1, INS-r3 and INS-r9. Octreotide and rapamycin induced a significant decrease in VEGF production by all three cell lines; LY294002 significantly inhibited VEGF production by STC-1 and INS-r3 only. We detected no effect of PD98059 whereas SB203850 significantly inhibited VEGF secretion in INS-r3 and INS-r9 cells only. By Western blotting analysis, we observed decreased intracellular levels of VEGF and HIF-1α under octreotide, rapamycin and LY294002. For rapamycin and LY294002, this effect was likely mediated by the inhibition of the mTOR/HIF-1/VEGF pathway. In addition to its well-known anti-secretory effects, octreotide may also act through the inhibition of the PI3K/Akt pathway, as suggested by the decrease in Akt phosphorylation detected in all three cell lines. In conclusion, our study points out to the complex regulation of VEGF synthesis and secretion in neoplastic GEP endocrine cells and suggests that the inhibition of VEGF production by octreotide and rapamycin may contribute to their therapeutic effects.

Localization of inhibins and activins in normal endocrine cells and endocrine tumors of the gut and pancreas: an immunohistochemical and in situ hybridization study.

Activins and inhibins, which belong to the TGF beta family, are composed of different combinations of alpha-, betaA-, and betaB-subunits, resulting in inhibin A (alphabetaA), inhibin B (alphabetaB), activin A (betaAbetaA), activin B (betaBbetaB), and activin AB (betaAbetaB). They regulate several cell functions, acting as paracrine/autocrine factors. Their actions, which depend on binding to specific receptors, are also modulated by follistatin. Gastroenteropancreatic (GEP) endocrine cells and endocrine tumors (ETs) produce several growth factors, but it is not well known whether they express follistatin and the various inhibin/activin subunits. We studied their expression in 65 GEP ETs using immunohistochemistry (IHC) and in situ hybridization (ISH). The alpha-subunit and follistatin were not identified in normal GEP endocrine cells and were poorly expressed in ETs. A betaA-subunit immunoreactivity (IR) was detected in A-, G-, EC-, and GIP-cells, while betaB-chain IR was present only in D-cells. The mRNAs encoding for these molecules were poorly expressed in normal tissues. BetaA- and betaB-subunits were identified in several ETs by both IHC and ISH: betaA-subunit mainly in G-cell and A-cell ETs, and betaB-subunit in D-cell, A-cell, and EC-cell ETs. Our results demonstrate a differential expression of activin/inhibin subunits among different types of GEP endocrine cells and related tumors, suggesting a role in modulation of biological functions of these normal and neoplastic endocrine cells.

Ghrelin in Fetal Thyroid and Follicular Tumors and Cell Lines: Expression and Effects on Tumor Growth

Ghrelin, a growth hormone-releasing hormone produced by gastroenteropancreatic endocrine cells, hypothalamus, and pituitary, was recently identified in medullary thyroid carcinomas and derived cell lines. However, no data exist on its expression in either normal or neoplastic thyroid follicular cells. We analyzed ghrelin expression by immunohistochemistry, in situ hybridization, and reverse transcriptase-polymerase chain reaction in 15 fetal, 4 infant, and 10 adult thyroids, and in 54 tumors of follicular origin. We also analyzed the effects of ghrelin on cell proliferation in N-PAP and ARO thyroid carcinoma cell lines. Ghrelin-binding sites were investigated using reverse transcriptase-polymerase chain reaction to detect its growth hormone secretagogue receptor (GHS-R) mRNA and an in situ-binding localization procedure. Strong ghrelin immunoreactivity was found in fetal but not in infant or adult thyroids. Ghrelin protein and mRNA were present, in variable amounts, in benign and malignant tumors. Normal thyroids, thyroid tumors, and cell lines showed ghrelin binding sites by binding localization, in the absence of the specific GHS receptor mRNA (with the exception of one normal thyroid). Moreover, ghrelin induced dose-dependent inhibition of growth in cell lines. In conclusion, ghrelin is expressed in fetal but not in adult thyroid, and is re-expressed in tumors; the presence of ghrelin receptors other than GHS-R in normal and neoplastic adult thyroid is suggested; ghrelin inhibits cell proliferation of thyroid carcinoma cell lines in vitro.

Ultrastructural classification of the endocrine cells of the large intestine of the calf. Cytochemical evidence of the presence of Viallis's pre-EC cells

GEP (Gastro-Entero-Pancreatic) endocrine cells were very numerous in the mucosal layer of the large intestine of the calf. Their frequence appeared to increase towards the distal portions of the gut. Endocrine cells were dispersed among epithelial cells lining intestinal glands and were frequently grouped together. Cellular shape was pyramidal or elongated; the cytoplasm was electron-lucent and contained highly characteristic secretory granules. Six different types of endocrine cells were identified on the basis of the ultrastructural aspect and cytochemical characteristics (silver-reactivity) of their secretory granules: EC, L, PP, A, D1 and D cells. EC and L cells were the most abundant in all localisations. They were especially numerous in the rectum. A subpopulation of EC cells was negative to Masson-Singh's reaction showing that they lack 5-HT. This observation enabled us to refer this latter cellular type to the "pre-EC" cells, described by Vialli as an earlier evolutive step of the EC cells population. Their presence in the calf gut might be linked to its possible "immaturity", due either to the age or to the alimentary diet.

Localization of insulin to gastroenteropancreatic cells in the turtle gastrointestinal tract

Insulin has been localized immunocytochemically to open-type gastroenteropancreatic endocrine cells in sections of Bouin's-fixed upper, middle, and lower intestine from Chrysemys picta, Pseudemys scripta scripta, P. scripta elegans, P. floridana, Sternotherus odoratus, and Trionyx spinifer asper. Radioimmunoassay of extracts of mucosal scrapings from Chrysemys intestine indicates differential amounts of insulin-like immunoreactivity within the intestine (higher amounts in the lower intestine) and in concentrations approximately one-tenth to one-fifth that found in extracts of Chrysemys pancreas. The presence of insulin in the gastrointestinal tract of chelonians represents a major departure from the typical vertebrate condition which is characterized by an absence of insulin from the spectrum of regulatory peptides in the gut.

Chromogranins in mammalian GEP endocrine cells: Their distribution and interrelations with co-stored amines and peptides.

Chromogranins (Cg) are large acidic proteins, co-stored with amines or peptides in the secretory granules of a wide variety of paraneurons. By immunohistochemistry performed on serial semithin sections, the distribution of Cg in the gastroenteropancreatic (GEP) system was analyzed in the endocrine pancreas of 10 mammalian species and in enteroendocrine cells of 6 species. Of the Cg families, CgA is primarily localized in GEP endocrine cells. CgA was regularly present in amine containing cells (EC and EC-like cells). In 11 other endocrine cell types, CgA-immunoreactivities showed inter-species and intra-species variations in their distribution or in the intensity of staining. However, no endocrine cell type lacked CgA-immunoreactivities in all species investigated. Findings from extended studies indicate that CgA is involved in the intracellular storage of resident amines, and in the storage of newly formed amines after exogenous application of the corresponding precursors. Finally, densitometric findings on possible reciprocal relationships between CgA- and peptide-immunoreactivities suggest that CgA also participates in the intragranular storage of peptide hormones in certain GEP endocrine cells.

Ontogeny of gastroenteropancreatic (GEP) endocrine cells in mouse and porcine embryos.

The ontogeny of the six main types of gastroenteropancreatic (GEP) endocrine cells was immunohistochemically studied in developing mouse and pig. Glucagon-immunoreactive cells first appeared in the duodenum and the primordial pancreas in 9-day-old mouse and 0.8 cm (18-day-old) porcine fetuses. In the mouse pancreas, following the appearance of the glucagon-immunoreactive cells, pancreatic polypeptide (PP)-, insulin-, gastrin/cholecystokinin (gastrin/CCK)- and somatostatin-immunoreactive cells were detected in 10-, 11-, 13- and 15- day-old fetuses. In the porcine pancreas, insulin-, somatostatin- and 5-hydroxytryptamine (5-HT)-immunoreactive cells were demonstrated in 1.0 cm (20-day-old) fetuses, while PP-immunoreactive cells were observed in 2.5 cm (30-day-old) fetuses. In contrast, in the mouse gastrointestinal tract, PP- and gastrin/CCK-immunoreactive cells were first found in 14-day-old fetuses, then 5-HT- and somatostatin-immunoreactive cells in 15- and 16-day-old fetused, respectively. In the porcine gut, four kinds of immunoreactive cells appeared in 2.0-3.0 cm (28-32 day-old) fetuses. It was demonstrated that insulin-immunoreactive cells were rarely seen in the porcine antrum and duodenum in the initial stage of development. These results indicate that the glucagon-immunoreactive cells first appear in the GEP endocrine system and that the endocrine cells in the primordial pancreas appear prior to those in the digestive tract proper. Therefore, it is suggested that the ontogenic patterns of GEP endocrine cells are basically similar in mice and pigs.

Distribution of chromogranin containing cells in the porcine gastroenteropancreatic endocrine system.

Regional distribution and ontogenetic time course of appearance of chromogranin (CG)-containing cells in the porcine gastroenteropancreatic (GEP) endocrine system were studied immunohistochemically. CG-immunoreactive cells were identified numerously in the gut and pancreas of adult pigs. In the overall gastrointestinal tract, they were most numerous in the fundic and pyloric gland regions of the stomach. In each portion of the gastrointestinal tract, the frequency of the CG-immunoreactive cells was higher than that of the total amount of any endocrine cells determined in our previous study. It was confirmed that all peptide/amine immunoreactive cell types except for pancreatic polypeptide-immunoreactive cells in the pancreas, and all argyrophil cell types detected by silver impregnation techniqucs, were immunoreactive with anti-CG serum. Ontogenetically, CG-immunoreactive cells had already appeared numerously in the primordial pancreas and moderately in the duodenum of 0.8 cm (18 days of gestation) porcine fetuses. In the pyloric antrum, the fundic portion and the remaining portion of the intestine, these cells appeared first in 1.5 cm (24-day-old), 3.0 cm (33-day-old) and 3.4 cm (35-day-old) fetuses, respectively. It was suggested that CG appeared much earlier than the proper peptide/amine hormones in the GEP endocrine cells at the initial stage of development. The possible significances of the early occurrence were also discussed.

Anti-lymphocyte antibody Leu-7 (HNK-1) recognizes a constituent of neuroendocrine granule matrix.

Anti-lymphocyte monoclonal antibody HNK-1 (Leu-7) reacts with the cell surfaces of natural killer (NK) lymphocytes and with myelin-associated glycoprotein (MAG). This antibody reacts intensely with normal and neoplastic adrenal medullary cells. A small proportion of normal pancreatic islet cells, anterior pituitary, and gastroenteropancreatic endocrine cells also show Leu-7 immunoreactivity. In adrenal medulla, ultrastructural immunocytochemical studies and immunoblot analyses reveal that Leu-7 reacts with an intracellular protein of MW 75 KD which is localized within the matrices of the chromaffin granules. The MW of this protein differs from those of MAG and chromogranin A. The findings suggest that Leu-7 immunoreactivity might be a new marker for specific subsets of secretory granules.

Immunohistochemical study of gastroenteropancreatic endocrine cells of the herbivorous Japanese field vole, Microtus montebelli

The gastroenteropancreatic (GEP) endocrine cells of the Japanese field vole were studied immunohistochemically. Somatostatin-, 5-hydroxytryptamine-, glicentin-, glucagon-, bovine pancreatic polypeptide-, gastrin-, gastric inhibitory polypeptide-, cholecystokinin-, substance P-, secretin-, neurotensin- and insulin-immunoreactive cells were revealed. The characteristic findings of the regional distribution and relative frequency of these immunoreactive cells in the GEP system of the vole were as follows. Somatostatin-immunoreactive cells were more numerous in the oxyntic glands than in the pyloric glands. Some somatostatin-immunoreactive cells were found in small clusters in the oxyntic glands. Gastrin-immunoreactive cells were detected not only in the pyloric glands and small intestine but also in the caecum and spiral colon. Gastric inhibitory polypeptide-immunoreactive cells were also detected in the pyloric glands and no motilin-immunoreactive cell was found in the gastroenteropancreatic system.

Immunocytochemical study of the gastroenteropancreatic endocrine cells of the sheep.

The gastroenteropancreatic (GEP) endocrine cells of the sheep were studied immunocytochemically and their distribution and frequency were determined. Eleven types of endocrine cells were revealed. In the abomasum, somatostatin-, gastrin-, glucagon- and glicentin-immunoreactive cells were detected with the highest frequency in the pyloric region. In the small intestine, somatostatin-, gastrin-, CCK-, motilin-, neurotensin-, secretin-, substance P-, glucagon-, glicentin- and BPP-immunoreactive cells were found and were most numerous in the duodenum except for neurotensin-, glucagon- and glicentin-immunoreactive cells which were more concentrated in the ileum. In the large intestine, somatostatin-, substance P-, glucagon-, glicentin- and BPP-immunoreactive cells were localized with the last three cell types being more concentrated in the rectum. In the pancreas, somatostatin-, glucagon-, glicentin-, BPP- and insulin-immunoreactive cells predominated within the islets and were also scattered in the exocrine portion and rarely detected in duct epithelial cells. The differences between the distribution and frequency of the GEP endocrine cells of the sheep and those of monogastric species are discussed.

Ultrastructure and immunohistochemistry of gastro-entero-pancreatic (GEP) endocrine cells in mucinous tumors of the ovary.

Twenty three mucinous tumors were studied by histochemical, auto‐fluorescence and immunoperoxidase methods, and electron microscopy to investigate the existence of gastro‐entero‐pancreatic (GEP) endocrine cells in mucinous tumors of the ovary. Out of 11 mucinous adenomas, 6 cases contained autofluorescence positive (EC‐) cells and 1 case contained somatostatin‐and gastrin‐cells. Out of 4 borderline malignancies, 4 cases contained EC‐cells and 3 cases contained somatostatin‐ and gastrin‐cells, and of 8 mucinous adenocarcinomas, 3 cases contained somatostatin‐cells and 2 cases contained EC‐ and gastrin‐cells. Electron microscopic observations of 7 cases including 4 mucinous adenomas, 2 borderline malignancies, and 1 adenocarcinoma revealed 3 different types of endocrine‐type granulated cells including EC‐, D‐and G‐cells. Furthermore, muco‐endocrine cell was found in a mucinous adenoma case. These findings agree with the results from autofluorescence and immunoperoxidase studies. The histogenetic origin of mucinous tumors is rather problematic and the appearance of various types of GEP endocrine cells support their endodermal derivation. These results strongly support the possibility that GEP endocrine cells in mucinous tumors are developed in loco from precursor endodermal cells.

The endocrine cells of the digestive system (author's transl)

Endocrine cells occur in the digestive system as micro-organs (islets of Langerhans) or scattered throughout the epithelium of the gastrointestinal tract ("diffuse endocrine epithelial organ" of Feyrter). These gastro-entero-pancreatic (GEP) endocrine cells synthesize--in addition to serotonin--a great variety of polypeptide hormones, which regulate both carbohydrate metabolism and digestive processes. The present review deals mainly with cytology and cytochemistry of GEP endocrine cells. A synopsis is presented of the 19 endocrine cell types identified to date, which includes their update nomenclature and their anatomical distribution pattern. Morphological-functional aspects of cell biology, pathology, and cytogenesis of theses cells and their position within superimposed systems (APUD cells, paraneurons) are discussed.

Immunoreactivities of gastrin (G-) cells. II. Non-specific binding of immunoglobulins to G-cells by ionic interactions.

Various gastro-entero-pancreatic (GEP) endocrine cells have been shown to contain concomitantly immunoreactivities against several peptide hormones. In the present study the "immunoreactivities" of gastrin (G-) cells of the rat stomach against 21 specific antisera and 10 control sera were investigated by means of the unlabelled antibody enzyme (PAP) technique using modifications of single steps in the immunocytochemical staining sequence. The results indicate that immunoglobulins can bind to gastrin cell granules obviously by non-specific ionic interactions. This non-specific binding of immunoglobulins occurs even in dilution ranges of the sera commonly used in immunohistochemical investigations of the GEP endocrine system. Since "adsorption controls" (preadsorption of the antisera with their respective antigens) will not discriminate between specific and non-specific binding of immunoglobulins to GEP endocrine cells additional specificity controls are necessary. In contrast to the immunostaining of various GEP endocrine cells by "established" antisera and of G-cells by gastrin antiserum immunoglobulins of sera from non-immunized animals as well as antibodies against corticotropin-lipotropin related peptides could be displaced from their binding sites in G-cells by alterations of the NaCl content of the buffers used as diluents or as rinsing solutions. To exclude immunostaining of GEP endocrine cells by nonspecific binding of immunoglobulins the following working procedures are recommended for immunocytochemical investigations of these cells: 1. Use of high titer antisera at low concentrations (diluted 1:1,500 or more). 2. Elevation of the salt (NaCl) content up to 0.5 M of the buffer used as diluent or as rinsing solution. 3. Adsorption controls will show reliable results only if point 1. and 2. have been taken into account.

Pathology of the neuroendocrine system.

Systematic use of immunocytochemistry and radioimmunoassay resulted in an enormous increase in the knowledge of the localization and secretion of peptides and amines. It is now known that many peptides and amines are present in the brain, in peripheral nerves, and in endocrine cells. The original APUD concept (8) had to be extended to include neural elements. At present it is realized that many activities of the body are regulated by this control system (currently called the neuroendocrine system). It’s messengers may act as neurotransmitters, as hormones, or as locally acting substances, the so-called paracrine substances. According to the type of secretion the neuroendocrine system may be considered to contain neurocrine (peptidergic neuron acting upon another neuron), neuroendocrine (neuron secreting its product(s) into the bloodstream), endocrine and paracrine cells. At present we are still at the very beginning of the unraveling of mechanisms resulting in disease of the neuroendocrine system. However, it is safe to predict that pathology of the system will be found to be complicated, and it is already now impossible to cover the subject in one workshop. Therefore, we selected 3 topics which may be of interest to biologists, physiologists, clinicians and pathologists, namely: “pathology of the pituitary;” “the concept and pathology of the disseminated (peripheral) neuroendocrine system;” and “pathology of the endocrine pancreas.” In these fields remarkable progress has been achieved in the understanding of pathophysiology and in accuracy of diagnosis. Immunocytochemistry and radioimmunoassay have significantly contributed to this progress and probably will change concepts of diseases of the neuroendocrine system. Part of this knowledge derived from retrospective analysis of routinely fixed operative samples and autopsy material. Recently, views of the biological behavior of pituitary tumors have dramatically changed. It is now obvious that the histologic classification based on granule staining properties is inadequate because the secretory potentialities of the tumors are far more extensive than is implied by this classification. Using immunocytochemistry it has been shown that so-called chromophobe adenomas often contain hormones (6) and that many pituitary tumors are multihormonal (4). Moreover, functional analysis by radioimmunoassay revealed that tumors often secrete more than one hormone. Hormonal secretion by pituitary tumors and tumor growth may also be stimulated or inhibited by drugs and functional tests and therefore can no longer be considered entirely autonomous (7). Together with the rapidly accumulating knowledge of hypothalamic regulation of pituitary hormone synthesis and secretion, such findings have led to a challenge of the concept of neoplasia (defined as “focal autonomous growth of tissue”). Pituitary “nodules” often behave as a focal hyperplasia (defined as “increase in the number ofcells”). The differentiation ofa tumor from hyperplasia is not merely of academic interest, it is of great importance to the neurosurgeon in prescribing treatment of the patient. The control device of the gastrointestinal tract is part of the disseminated neuroendocrine system. The gastroenteropancreatic peptides are localized in single cells and/or in neuronal cell bodies and their axons. The widespread distribution of these peptides produces an integrated response to diffuse and discontinuous stimuli involved in the intake of food, its transport along the gastrointestinal tract, its digestion and resorption and in the flow of metabolic fuel ( 1). The triple control device of the neuroendocrine system, endocrine, paracrine and neural control (9), can be especially well demonstrated in the gastrointestinal tract. The endocrine pancreas is part of the system of gastroenteropancreatic endocrine cells. The knowledge of its structure and physiology

Mechanisms of release of gastro-entero-pancreatic endocrine cells.

AbstractEndocrine cells of the gastro‐entero‐pancreatic (GEP) system release their hormones in response to nutrient stimulation. Gastro‐entero endocrine cells receive stimulation from the circulation and lumen and have the potential to secrete into the circulation and lumen. Pancreatic endocrine cells cannot receive stimulation from nor secrete into the lumen.Antral gastrin‐producing G cells released hormone in a biphasic manner in response to feeding; the initial plasma gastrin peak was absent after a 48‐hour fast. The increase of antral tissue gastrin detected was far more than that required for the major plasma gastrin response. Intravenous hyperalimentation (IVH) (30% dextrose, 12% amino acids) was used to assess the effect of the absence of luminal stimulation upon GEP hormone (gastrin, gastric inhibitory polypeptide, glucagon, and insulin) release. During the first 6 days of IVH, circulating insulin concentrations increased absolutely and in relationship to glucagon; gastrin concentrations decreased to undetectable levels. During the following 4 days of IVH, circulating insulin decreased and gastrin increased to their initial concentrations. The IVH responses reflect the abilities of the different cells to adapt to intravenous nutrients.GEP endocrine cells have distinct differences in their hormone turnover mechanisms in response to nutrient stimulation. These different cellular response mechanisms may provide a clue to the functional evolution and sophistication of GEP endocrine cells.