Conditional deletion of menin results in antral G cell hyperplasia and hypergastrinemia

Antral gastrin is the hormone known to stimulate acid secretion and proliferation of the gastric corpus epithelium. Patients with mutations in the multiple endocrine neoplasia type 1 (MEN1) locus, which encodes the protein menin, develop pituitary hyperplasia, insulinomas, and gastrinomas in the duodenum. We previously hypothesized that loss of menin leads to derepression of the gastrin gene and hypergastrinemia. Indeed, we show that menin represses JunD induction of gastrin in vitro. Therefore, we examined whether conditional deletion of Men1 (Villin-Cre and Lgr5-EGFP-IRES-CreERT2), with subsequent loss of menin from the gastrointestinal epithelium, increases gastrin expression. We found that epithelium-specific deletion of Men1 using Villin-Cre increased plasma gastrin, antral G cell numbers, and gastrin expression in the antrum, but not the duodenum. Moreover, the mice were hypochlorhydric by 12 mo of age, and gastric somatostatin mRNA levels were reduced. However, duodenal mRNA levels of the cyclin-dependent kinase inhibitor p27(Kip1) were decreased, and cell proliferation determined by Ki67 staining was increased. About 11% of the menin-deficient mice developed antral tumors that were negative for gastrin; however, gastrinomas were not observed, even at 12 mo of age. No gastrinomas were observed with conditional deletion of Men1 in the Lgr5 stem cells 5 mo after Cre induction. In summary, epithelium-specific deletion of the Men1 locus resulted in hypergastrinemia due to antral G cell hyperplasia and a hyperproliferative epithelium, but no gastrinomas. This result suggests that additional mutations in gene targets other than the Men1 locus are required to produce gastrin-secreting tumors.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry.

Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry

Summary Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Ultrastructural localization of chromogranin: a potential marker for the electron microscopical recognition of endocrine cell secretory granules

Summary Using a monoclonal antibody (LK2H10) directed against human chromogranin, we have been able to localize this soluble glycoprotein to the matrix of secretory granules from a wide variety of endocrine cells. In the gut, enterochromaffin, enteroglucagon, glucose-dependent insulinotropic peptide, gastrin, and neurotensin-containing cells exhibit chromogranin immunoreactivity. In our system, chromogranin-immunoreactive material was restricted to the halo of human pancreatic glucagon-containing secretory granules within A-cells. Chromogranin immunoreactivity was also localized to secretory granules in phaeochromocytomas, gastrinomas, medullary carcinomas of the thyroid and a carotid body tumour (chemodectoma). Chromogranin is proposed as a potential marker for the ultrastructural recognition of endocrine cell secretory granules.     

Growth hormone-like immunoreactivity in gastrin cells and gastrinomas.

Growth hormone (GH)-immunoreactive material was found to occur in the antral gastrin cells and in scattered cells of the pancreatic islets in several mammalian species, including man. Examination of gastrinomas revealed the majority of tumour cells to display GH-like immunoreactivity.     

Combined non-radioactive detection of peptide hormones and their mRNA's in endocrine cells.

Non-radioactive in situ hybridization employing detection of biotin-labeled probes by an alkaline phosphatase-based procedure has proven useful for demonstrating a wide variety of mRNA species. With certain developers, the alkaline phosphatase reaction product is both light microscopically visible and fluorescent. We have exploited this to perform simple double-stainings for mRNA's and their corresponding peptide products in human insulin and gastrin cells and in rat ACTH, MSH and gastrin cells. Such stainings show that nearly all of these cells simultaneously contain both the peptide hormone and its corresponding mRNA. Human gastrin cells show a differentiated localization of gastrin mRNA and gastrin. Thus, while gastrin immunofluorescence predominates in secretory granules present in the basolateral region of the cells, gastrin mRNA is virtually restricted to the supranuclear region of the cells. Here it may be preferentially associated with granular endoplasmic reticulum. The strict subcellular localization of gastrin mRNA differs from that of general polyadenylated RNA in the G cells and raises questions whether specific transport routes or sites of accumulation for defined mRNA species exist.     

Loss of heterozygosity on chromosome 11 in sporadic gastrinomas

Summary Gastrinomas are pancreatic endocrine neoplasms that arise either sporadically or are inherited as part of the multiple endocrine neoplasia type I syndrome (MEN I). Loss of heterozygosity (LOH) in the region flanking the MEN I gene at chromosome 11q13 has been documented in a few sporadic and familial pancreatic endocrine tumors, but not previously in sporadic gastrinomas. It has therefore been suggested that gastrinomas develop by a mechanism different from other tumors associated with the MENI syndsrome. We report LOH on chromosome 11 in 5 of 11 sporadic gastrinomas. Four of these tumors have LOH for markers flanking the MEN I region. Molecular evaluation of segments of chromosomes 3, 13, and 17 known to contain cloned or putative tumor suppressor genes fail to show LOH except at one locus in one tumor. These data suggest that a tumor suppressor DNA segment exists at 11q13 that may be involved in the development of sporadic gastrinomas.     

Simultaneous localization of calcitonin mRNA and peptide in a medullary thyroid carcinoma

We report the visualization of calcitonin gene expression products at the mRNA and peptide levels on the same section of a medullary thyroid carcinoma by combined in situ hybridization and immunohistochemistry. mRNA detection was accomplished by hybridization with radioactively labeled antisense RNA probes followed by autoradiography and immunohistochemically using the avidin-biotin complex method. Best results were obtained when in situ hybridization preceded immunohistochemistry, as determined by quantitative analysis of the autoradiographs. When immunohistochemistry was performed prior to in situ hybridization, the RNase inhibitor heparin had to be added to the antibodies to retain hybridizable mRNA. The intensity of the two reactions varied in individual cells, indicating a functional heterogeneity of tumor cells with regard to calcitonin mRNA content and storage of the related immunoreactive peptide. These results, in combination with elevated serum calcitonin levels, suggest significant differences in the rate of secretion of individual tumor cells. Simultaneous localization of mRNA and its peptide within the same cell may, therefore, provide further insight into gene expression and secretory activity at the single cell level.     

Combined non-radioactive detection of peptide hormones and their mRNA's in endocrine cells

Summary Non-radioactive in situ hybridization employing detection of biotin-labeled probes by an alkaline phosphatase-based procedure has proven useful for demonstrating a wide variety of mRNA species. With certain developers, the alkaline phosphatase reaction product is both light microscopically visible and fluorescent. We have exploited this to perform simple double-stainings for mRNA's and their corresponding peptide products in human insulin and gastrin cells and in rat ACTH, MSH and gastrin cells. Such stainings show that nearly all of these cells simultaneously contain both the peptide hormone and its corresponding mRNA. Human gastrin cells show a differentiated localization of gastrin mRNA and gastrin. Thus, while gastrin immunofluorescence predominates in secretory granules present in the basolateral region of the cells, gastrin mRNA is virtually restricted to the supranuclear region of the cells. Here it may be preferentially associated with granular endoplasmic reticulum. The strict subcellular localization of gastrin mRNA differs from that of general polyadenylated RNA in the G cells and raises questions whether specific transport routes or sites of accumulation for defined mRNA species exist.     

Ultrastructural studies of endocrine cell populations showing an argentaffin reaction and/or serotonin immunoreactivity in the rat antral mucosa

Summary On the basis of staining results in closely related semi-thin sections from rat antral mucosa immunostained with polyclonal serotonin antibodies and silver-stained for the argentaffin reaction, respectively, three different cell populations could be distinguished. One of these cell populations showed both serotonin immunoreactivity and an argentaffin reaction, a second one serotonin immunoreactivity alone, and a third one only an argentaffin reaction.These cell populations were studied electron microscopically in ultra-thin sections located between the stained semi-thin sections. The cell population displaying an agentaffin reaction and serotonin immunoreactivity showed secretory granules of the enterochromaffin cell type. A similar granular appearance was observed in cells which only exhibited an argentaffin reaction. Serotonin immunoreactivity in the absence of an argentaffin reaction was evident in some G (gastrin) cells. and in some D₁ and possibly also some D (somatostatin) cells; but not all the endocrine cells of the non-enterochromaffin type displayed serotonin immunoreactivity. The significance of the different reactions in the three cell populations is discussed.     

Gastrin and ACTH-like immunoreactivity occurs in two ultrastructurally distinct cell types of rat antropyloric mucosa

Summary The properties of endocrine cells of rat antropyloric mucosa, which simultaneously store both gastrin and ACTH-like immunoreactivity have been examined. In freely fed animals all or nearly all antral gastrin cells contain also large quantities of ACTH-like immunoreactivity. Following three days of fasting the gastrin cell content of ACTH-like peptides is drastically reduced, but increases rapidly upon refeeding of the starved animals for 30 min. At the electron microscopical level, the vast majority of cells storing both gastrin and ACTH-like peptides are identified as G cells but, in addition, a few, previously unrecognized, endocrine cells have also been found to store both types of peptides. The latter new cell type has tentatively been labelled the G_a cell. In normal freely fed animals the G cell is characterized by the occurrence of both electron-dense and electron-lucent granules. Correlative immunocytochemical and ultrastructural studies indicate that gastrin and the ACTH-like peptides are both stored in the cytoplasmic granules. Our results indicate that the gastrin cells release their content of ACTH-like peptides in response to fasting and that this release is blocked by refeeding. The differential release of two hormone-like substances from the same endocrine cell type is of great interest for analysis of mechanisms of peptide hormone release.     

Immunohistochemical localization of bombesin-like peptides in the digestive tract of the bowfin,Amia calva

1. The distribution of bombesin-like immunoreactivity was determined in the gastrointestinal tract of the primitive holostean fish, the bowfin (Amia calva) using immunocytochemistry.2. Immunostaining using two different antisera raised against frog skin bombesin revealed a population of apparent endocrine cells containing bombesin-like immunoreactivity in the epithelium of the antral mucosa in the stomach.3. No bombesin-containing endocrine cells were present in any other segment of the gut. Bombesinergic nerves were not observed anywhere in the bowfin gastrointestinal tract.4. The antral bombesin endocrine cells were of the open type and were distributed diffusely from the base to the tips of antral glands, with some tendency to cluster near the base of the glands.5. These results suggest that a bombesin-like peptide may play an endocrine role in control of gastric functions such as regulation of acid secretion and gastric motility. These results support the hypothesis that bombesin serves a role in bony fish analogous to the role of antral cholecystokinin in amphibia and antral gastrin in amniotes.     

Vesicular monoamine transporters in the rat stomach.

Cellular distribution of vesicular monoamine transporters (VMATs), known to regulate vesicular storage and release of biogenic amines (i.e., catecholamines, serotonin, histamine, etc.), have been studied in the rat stomach using in situ hybridization histochemistry (ISHH) and immunohistochemical (IHC) techniques. 35S-UTP labeled riboprobes showed that mRNAs of both VMATs are expressed in the gastric mucosa. A combination of ISHH and IHC verified that most of the parietal cells (among other epithelial cells) express mRNA of the peripheral type transporter (VMAT1) while enterochromaffin-like cells (ECL) of the fundic mucosa express mRNA of the central type (VMAT2). In addition, with double fluorescent IHC we detected VMAT1 protein in serotoninergic enterochromaffin cells (EC) of the stomach and in gastrin producing G cells of the antral mucosa. Similarly to the fundus, VMAT2 protein was present in ECL cells and in the enteric plexus. Surprisingly, serotonin- and/or histamine-containing cells in the connective tissue compartments of the stomach (i.e., lamina propria and submucosa), immunoreactive for a mast cell specific antigen, displayed neither VMATI nor VMAT2 immunoreactivity. Distribution of VMATs in the rat stomach support our previous observations on aminergic properties of two important gastrointestinal (GI) epithelial cell populations primarily known for other specific secretory products, i.e. dopaminergic properties of acid producing parietal cells and histaminergic properties of gastrin producing G cells. These data emphasize the existence of a non-neuronal, intrinsic aminergic system in the GI tract.     

In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors.

The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with 35S-labeled oligonucleotide probes in formalin-fixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the mRNA for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of mRNA expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.     

Growth hormone-like immunoreactivity in gastrin cells and gastrinomas

Summary Growth hormone (GH)-immunoreactive material was found to occur in the antral gastrin cells and in scattered cells of the pancreatic islets in several mammalian species, including man. Examination of gastrinomas revealed the majority of tumour cells to display GH-like immunoreactivity.The present findings were first reported at the 2nd International Symposium on Gastrointestinal Hormones in Beito, Norway, August 1978. Abstract: R. Håkanson, J. Alumets, F. Sundler: Scand. J. Gastroenterol.13, Suppl. 49, p. 75 (1978)     

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.     

Somatostatin cells in rat antral mucosa: qualitative and quantitative ultrastructural analyses in different states of gastric acid secretion

Summary In the gastrointestinal tract somatostatin is localized in endocrine cells and in neurons. The antral somatostatin (D-) cell shares features of both cell types. The activity of the antral D-cell is regulated by intragastric pH. Therefore different states of gastric acidity were induced experimentally in order to study D-cell morphology at the electron microscopical level. The morphological findings were related to measurements of plasma and tissue concentrations of the peptide. The D-cell is characterized by extensive membrane interdigitations with neighbouring cells. Changes in the activity of antral D-cells are reflected by an increase in cytoplasmic secretory granule density and a shift of secretory granules towards basal cell processes. Direct endocrine cell contacts at the level of the perikarya were rarely observed. The intracellular distribution of secretory granules suggests that cell communication is more likely to take place at the level of the strongly immunoreactive cytoplasmic processes. No evidence for endocrine or exocrine (luminar) secretion was observed morphologically. This is in agreement with the concept of paracrine secretion of the antral D-cell.     

ACTH-like immunoreactivity in the gastrin cell. Independent changes in gastrin and ACTH-like immunoreactivity during ontogeny.

Rat antral gastrin cells have been shown to contain ACTH-like immunoreactivity. Studies on the ontogeny of the antral gastrin cells reveal that these cells start to store gastrin before they contain detectable quantities of ACTH-like immunoreactivity. At no stage studied were duodenal gastrin cells found to contain ACTH-like peptides. The data indicate that the G cells synthetizes and/or releases the two hormonal peptides independently.