Endocrine Cells In Mouse Research Articles

The Impact of Different Implantation Sites and Sex on the Differentiation of Human Pancreatic Endoderm Cells Into Insulin-Secreting Cells InVivo.

Few studies have directly compared the differentiation of human embryonic stem cell-derived progenitors in different implantation sites in male and female recipients. We investigated whether the site of implantation and/or the sex of the recipient influenced the differentiation of pancreatic progenitors invivo in mice. Mice implanted with cells in macroencapsulation devices contained fewer off-target structures and developed stimulated insulin release faster than other implant sites, while females implanted with cells under the kidney capsule developed stimulated insulin release before males. Macroencapsulation devices reduced the formation of off-target cells from human embryonic stem cell-derived progenitors, a useful characteristic for clinical applications.

Gastric tissue makes insulin

Reprogramming Diabetics lack insulin-producing pancreatic beta cells. One therapeutic strategy involves generating these cells from other sources and then transplanting them into patients. To withstand the pathological diabetic environment, a renewable source of beta cells is crucial. Ariyachet et al. demonstrate that stomach cells fit the bill. Like the pancreas, the stomach arises from the endoderm and harbors many stem and progenitor cells. Introducing the trio of proteins into mouse endocrine cells from the lower stomach reprograms them into insulin-producing beta cells. Transplanting reprogrammed stomach mini-organs into diabetic mice allowed them to control their hyperglycemia, suggesting a viable gastric source for the production of insulin. Cell Stem Cell 10.1016/j.stem.2016.01.003 (2016).

Effects of unilateral cervical vagotomy on antral endocrine cells in mouse.

The present study was carried out to investigate the effect of unilateral cervical vagotomy on the antral endocrine cells in mouse. Fifty-four mice were randomly divided into three groups, 18 in each, for left or right cervical vagotomy, or sham operation as controls. The animals were sacrificed 2, 4, and 8 weeks after the operation, respectively. Chromogranin-, gastrin/CCK-, serotonin-, and somatostatin-cells were detected by immunohistochemistry and quantitated by computerised image analysis. The results showed that the number of chromogranin-cells was decreased in both left and right vagotomized mice after 4 weeks and remained at the same level after 8 weeks. The numbers of gastrin-, serotonin- and somatostatin-cells did not change after right vagotomy. However, the numbers of gastrin- and somatostatin-cells were decreased after left vagotomy, whereas no change was found in serotonin-cells. Endocrine cells with vacuolated cytoplasm and pyknotic nuclei were also observed during the course of time. The alteration in the antral endocrine cells observed in this study seemed to be dynamic and depended on the observation time after the operation as well as the denervated branches of the vagus nerve. This may explain, at least partially the contradictory results obtained earlier by different investigators.

Changes in intestinal endocrine cells in the mouse after unilateral cervical vagotomy.

The effect of right or left unilateral cervical vagotomy on the intestinal endocrine cells was studied in 23 mice at 2 and 8 weeks after operation, respectively. The results were compared with that from 10 sham operated mice. Various types of endocrine cells in duodenum and proximal colon were detected by immunohistochemistry and quantified by computerized image analysis. In mouse duodenum, chromogranin-, CCK/gastrin-, GIP- and somatostatin-cells were significantly decreased at 2 weeks after right vagotomy, but returned to the control levels at 8 weeks. Serotonin-cells were reduced at both 2 and 8 weeks after right vagotomy. The amount of the duodenal endocrine cells did not change after left vagotomy with the exception of secretin-cells, which were diminished at 8 weeks after both right and left vagotomy. In the proximal colon, chromogranin-cells were also decreased at 2 weeks after right vagotomy. Serotonin-cells were reduced at 8 weeks after left vagotomy but not right vagotomy. There was no significant difference between the unilaterally vagotomized and the sham operated mice with regard to PYY- and glucagon-cells. It was concluded that vagotomy affected the intestinal endocrine cells in mouse. The influence was more pronounced in the small intestine than the proximal colon. The right vagus nerves seemed to exert more effect on the intestinal endocrine cells than the left ones.

Widespread expression of zinc transporter ZnT (SLC30) family members in mouse endocrine cells

Zinc is abundant in most endocrine cell types, and plays a pivotal role in the synthesis and secretion of many hormones. Recent studies have demonstrated the expression of numerous zinc transporter (ZnT) family members in the pancreas, thyroid, and adrenal glands, suggesting a role for ZnTs in regulating cellular zinc homeostasis in endocrine cells. However, the cellular distribution of ZnTs in the endocrine organs has not been well established. In the present study, the mRNA expression level, cellular distribution of ZnTs as well as liable zinc ions were examined in the mouse pituitary, adrenal glands, thyroid, and pancreas. In general, ZnT1-10 mRNA was expressed to various degrees in the detected endocrine organs, with no detectable ZnT10 mRNA in the pancreas. In the anterior pituitary, both the acidophilic and basophilic cells were immunopositive to ZnT1-5, 7, 8, except for ZnT10. In the adrenal cortex, the immunoreactivity of all the tested ZnTs, including ZnT1-5, 7, 8, 10, was observed in the zona fasciculata, and some ZnTs were detected in the zona glomerulosa, zona reticularis, and the adrenal medulla. Both the follicle epithelial cells and parafollicular cells in the thyroid gland were immunostained with ZnT1-5, 7, 8, but not ZnT10. In the endocrine pancreas, the immunoreactivity of tested ZnTs was observed to various degrees except for ZnT10 in the cytoplasm of islet cells. Furthermore, autometallographic staining showed that liable zinc was observed in the endocrine cells, such as the adrenal cortical cells, thyroid follicle epithelial cells, and the pancreatic islet cells. All together, the wide distribution of liable zinc and the phenomenon that numerous ZnT family members are partially overlapped in a subset of endocrine cells suggest an important role for the ZnT family in controlling cellular zinc levels and subsequently regulating the synthesis and secretion of hormones in the endocrine system.

Innervation Patterns of Autonomic Axons in the Human Endocrine Pancreas

The autonomic nervous system regulates hormone secretion from the endocrine pancreas, the islets of Langerhans, thus impacting glucose metabolism. The parasympathetic and sympathetic nerves innervate the pancreatic islet, but the precise innervation patterns are unknown, particularly in human. Here we demonstrate that the innervation of human islets is different from that of mouse islets and does not conform to existing models of autonomic control of islet function. By visualizing axons in three dimensions and quantifying axonal densities and contacts within pancreatic islets, we found that, unlike mouse endocrine cells, human endocrine cells are sparsely contacted by autonomic axons. Few parasympathetic cholinergic axons penetrate the human islet, and the invading sympathetic fibers preferentially innervate smooth muscle cells of blood vessels located within the islet. Thus, rather than modulating endocrine cell function directly, sympathetic nerves may regulate hormone secretion in human islets by controlling local blood flow or by acting on islet regions located downstream.

Identification of the ectonucleotidases expressed in mouse, rat, and human Langerhans islets: potential role of NTPDase3 in insulin secretion

Extracellular nucleotides and adenosine regulate endocrine pancreatic functions such as insulin secretion by Langerhans islet β-cells via the activation of specific P2 and P1 receptors. Membrane-bound ectonucleotidases regulate the local concentration of these ligands and consequently control the activation of their receptors. The objective of this study was to identify and localize the major ectonucleotidases, namely NTPDases and ecto-5'-nucleotidase, present in the endocrine pancreas. In addition, the potential implication of ecto-ATPase activity on insulin secretion was investigated in the rat β-cell line INS-1 (832/13). The localization of ectonucleotidase activity and protein was carried out in situ by enzyme histochemistry and immunolocalization in mouse, rat, and human pancreas sections. NTPDase1 was localized in all blood vessels and acini, and NTPDase2 was localized in capillaries of Langerhans islets and in peripheral conjunctive tissue, whereas NTPDase3 was detected in all Langerhans islet cell types. Interestingly, among the mammalian species tested, ecto-5'-nucleotidase was present only in rat Langerhans islet cells, where it was coexpressed with NTPDase3. Notably, the inhibition of NTPDase3 activity by BG0136 and NF279 facilitated insulin release from INS-1 (832/13) cells under conditions of low glycemia, probably by affecting P2 receptor activation. NTPDase3 activity also regulated the inhibitory effect of exogenous ATP in the presence of a high glucose concentration most likely by controlling adenosine production. In conclusion, all pancreatic endocrine cells express NTPDase3 that was shown to modulate insulin secretion in rat INS-1 (832/13) β-cells. Ecto-5'-nucleotidase is expressed in rat Langerhans islet cells but absent in human and mouse endocrine cells.

Pancreatic Islet Immunoreactivity to the Reg Protein INGAP

The Reg-related protein family member INGAP (islet neogenesis-associated protein) is a pleiotropic factor enhancing islet neogenesis, neurite growth, beta-cell protection, and beta-cell function. Using an antibody to the N-termini of INGAP, we have identified that immunoreactivity to INGAP localized to the pancreatic endocrine cells in mouse. INGAP- and insulin-immunoreactive cells are mutually exclusive, with INGAP-immunoreactive cells being preserved after streptozotocin-mediated destruction of beta-cells. Glucagon- and INGAP-immunoreactive cells colocalize, although respective antigen expression occurs in different intracellular locations. These data suggest that INGAP-immunoreactive cells include alpha-cells; however, detection of single INGAP-immunoreactive/glucagon-negative cells indicates that this may not be exclusive. In addition to mouse, detection of islet endocrine cells that were INGAP immunoreactive/glucagon immunoreactive/insulin negative was also observed in islets from human, monkey, and rat. These findings reveal that INGAP and/or related group 3 Reg proteins have a conserved expression in the pancreatic islet.

Preservation of proliferating pancreatic progenitor cells by Delta-Notch signaling in the embryonic chicken pancreas

BackgroundGenetic studies have shown that formation of pancreatic endocrine cells in mice is dependent on the cell autonomous action of the bHLH transcription factor Neurogenin3 and that the extent and timing of endocrine differentiation is controlled by Notch signaling. To further understand the mechanism by which Notch exerts this function, we have investigated pancreatic endocrine development in chicken embryos.ResultsIn situ hybridization showed that expression of Notch signaling components and pro-endocrine bHLH factors is conserved to a large degree between chicken and mouse. Cell autonomous inhibition of Notch signal reception results in significantly increased endocrine differentiation demonstrating that these early progenitors are prevented from differentiating by ongoing Notch signaling. Conversely, activated Notch1 induces Hes5-1 expression and prevents endocrine development. Notably, activated Notch also prevents Ngn3-mediated induction of a number of downstream targets including NeuroD, Hes6-1, and MyT1 suggesting that Notch may act to inhibit both Ngn3 gene expression and protein function. Activated Notch1 could also block endocrine development and gene expression induced by NeuroD. Nevertheless, Ngn3- and NeuroD-induced delamination of endodermal cells was insensitive to activated Notch under these conditions. Finally, we show that Myt1 can partially overcome the repressive effect of activated Notch on endocrine gene expression.ConclusionWe conclude that pancreatic endocrine development in the chicken relies on a conserved bHLH cascade under inhibitory control of Notch signaling. This lays the ground for further studies that take advantage of the ease at which chicken embryos can be manipulated.Our results also demonstrate that Notch can repress Ngn3 and NeuroD protein function and stimulate progenitor proliferation. To determine whether Notch in fact does act in Ngn3-expressing cells in vivo will require further studies relying on conditional mutagenesis.Lastly, our results demonstrate that expression of differentiation markers can be uncoupled from the process of delamination of differentiating cells from the epithelium.

Context-dependent regulation of NeuroD activity and protein accumulation

NeuroD/BETA2 (referred to as NeuroD hereafter) is a basic helix–loop–helix (bHLH) transcription factor that is required for the development and survival of a subset of neurons and pancreatic endocrine cells in mice. Gain-of-function analyses demonstrated that NeuroD can (i) convert epidermal fate into neuronal fate when overexpressed in Xenopus embryos, and (ii) activate the insulin promoter in pancreatic beta cell lines in response to glucose stimulation. In glucose-stimulated INS-1 pancreatic beta cells, mutations of S259, S266, and S274 to alanines inhibited the ability of NeuroD to activate the insulin promoter. Phosphorylation of those serine residues by ERK1/2 was required for NeuroD activity in that assay. To determine whether the same residues are implicated in the neurogenic activity of NeuroD, we mutated the conserved S259, S266, and S274 of Xenopus NeuroD to alanines (S259A, S266A, and S274A), and performed an ectopic neurogenesis assay in Xenopus embryos. In contrast to what has been observed in the pancreatic beta cell line, the S266A and S274A mutant forms of Xenopus NeuroD displayed significantly increased abilities to form ectopic neurons, while S259A had little effect. In addition, S266A and S274A of Xenopus NeuroD resulted in increased accumulation of protein in the injected embryos while the corresponding mutations on mouse NeuroD did not have the same effect in an insulinoma cell line. Our results demonstrate that the consequence of NeuroD protein modification is context-dependent at both the molecular and functional levels.

Duodenal endocrine cells in mice with particular regard to age-induced changes.

Duodenal endocrine cell types in four age groups of NMRI mice (1, 3, 12 and 24 months old) were identified by immunocytochemistry and quantified by computerized image analysis. Whereas the number of secretin-immunoreactive cells was significantly increased in the 24-month-old group, the number of GIP-immunoreactive cells was reduced in 12-month-old compared with 3-month-old mice. The number of somatostatin-immunoreactive cells was fewer in both the 12- and 24-month-olds vis-à-vis the 3-month-old mice. Whereas serotonin-immunoreactive cells were fewer in both 1-month-old and 12-month-old mice, they were more numerous in 24-month-old mice then in the 3-month-old ones. The number of gastrin/CCK-immunoreactive cells was unaffected by age. The cell secretory index (CSI) of secretin- and serotonin-immunoreactive cells was increased in the 24-month-old mice vis-à-vis the 3-month-old ones and the CSI of GIP- and somatostatin-immunoreactive cells was increased in 12-month-old mice vis-à-vis 3-month-old mice. In contrast, the CSI of somatostatin- and serotonin-immunoreactive cells in 1-month-old mice was lower than that of 3-month-old-mice. The nuclear volume of secretin-, GIP-, gastrin/CCK- and serotonin-immunoreactive cells was less in 1-month-olds than in 3-month-old mice. Whereas the nuclear volume of somatostatin-immunoreactive cells was decreased in 12-month-old animals, that of gastrin/CCK- and serotonin-immunoreactive cells was greater in 24-month-old mice than in 3-month-old ones. It is concluded that these changes may be secondary to structural and functional changes in the gastrointestinal tract caused by ageing. It is possible that these changes are involved in the development of dysfunction of the gut observed at advanced age.

Age-related changes in antral endocrine cells in mice.

Antral endocrine cells in four age groups of mice, namely prepubertal (1 month old), young (3 months old), ageing (12 months old) and senescent (24 months old), were detected by immunocytochemistry and quantified by computerized image analysis. A statistical difference was detected between the different age groups regarding the numbers of gastrin-, somatostatin-, and serotonin-immunoreactive cells. The number of gastrin-immunoreactive cells significantly increased between 1 and 12 months, whereas they became significantly fewer between 12 and 24 months. Somatostatin-immunoreactive cell number increased significantly in 1-, 12- and 24-month-old mice, compared with young mice (3 months old). The number of serotonin-immunoreactive cells also increased significantly in 1- and 12-month-old mice as compared with young mice. There was a statistical difference between different age-groups regarding the cell secretory index (CSI) of somatostatin- and gastrin-immunoreactive cells, the CSI of both somatostatin- and serotonin-immunoreactive cells increased significantly in 1-, 12-, and 24-month-old mice, compared with young mice. There was no statistical difference between the different age-groups regarding the CSI of gastrin-immunoreactive cells, nor between males and females regarding the number and CSI of all the endocrine cell types investigated. It is suggested that the large number of somatostatin-immunoreactive cells in ageing and senescent mice might have an impact on the gastric delay seen in the elderly. It was concluded also that the changes in the antral endocrine cells could be involved in the development of dysfunction of the gastrointestinal tract inherent in ageing, or could be secondary to structural and functional changes in the alimentary tract caused by ageing.

Metallothionein does not protect mouse endocrine cells from damage induced by alloxan injection

Effects of metallothionein (MT) on pancreatic endocrine cells of mice, injected with alloxan and at different zinc status were studied. Mice were given drinking water containing four different concentrations of zinc (0, 0.05, 0.1 or 0.5%) for 18 days, and alloxan was injected once on the 14th day. When zinc was added to the drinking water, pancreatic zinc and MT contents increased after injection of alloxan, but did not change with injection of vehicle alone, except in the group of mice drinking 0.5% zinc in water. However, plasma glucose level was increased in all the alloxan injected groups, and was independent of their zinc status. In mice given water with 0.5% of zinc, both pancreatic zinc and MT contents were higher than control mice given water alone. There was no difference in zinc and MT contents of the pancreas in mice drinking 0.5% zinc in groups injected with either alloxan or vehicle. The increase in plasma α-amylase activity, an indicator of pancreatic exocrine toxicity, was observed only in mice drinking water with 0.5% zinc after injection of both alloxan and vehicle. Histochemically, degranulation of zymogen and duct-like structures of exocrine cells and atrophy and disappearance of islet cells were observed in alloxan-injected mice drinking 0.5% zinc in water. The zymogen degranulation was observed on the vehicle-injected mice drinking 0.5% zinc in water. MT was immunohistochemically detected in the exocrine cells of both alloxan- and vehicle-injected mice given 0.5% zinc in water. No MT was detected in islet cells of mice in any group. The results show that an increase of zinc content may be followed by induction of MT synthesis in the pancreas of mice given increasing amounts of zinc in drinking water. However, MT dose not provide any protection against damage caused by alloxan to endocrine cells of the pancreas.

Image Analysis of the Duodenal Endocrine Cells in Mice with Particular Regard to Optical Densitometry

The endocrine cells in the murine proximal duodenum have been investigated by means of immunohistochemistry and computerized image analysis. Five endocrine cell types were identified, namely secretin-, gastric inhibitory polypeptide (GIP)-, gastrin-CCK-, somatostatin- and serotonin immunoreactive cells. The number of endocrine cells/mm3 epithelial cells was estimated and the cell secretory index (CSI) for different endocrine cell types was determined. Furthermore, the optical density of the cellular immunoreactivity and the immunoreactive area in the cell were determined and an index, cell immunoreactivity content was estimated as the optical density multiplied by the immunoreactive area. It has been suggested that the use of this index might better reflect the cellular peptide/amine content than does the CSI. Serotonin-immunoreactive cells were the predominant endocrine cell type, followed by gastrin/CCK-immunoreactive cells. The numbers of secretin-, GIP- and somatostatin immunoreactive cells were almost identical. All endocrine cell types were present both in crypts and in villi, but were, more numerous in the crypts, except for secretin which was more frequent in the villi.

Morphological characteristics of intestinal endocrine cells in mice

Morphological characteristics of intestinal endocrine cells in mice

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 and development of peptidergic nerves and gut endocrine cells in mice with congenital aganglionic colon, and their normal littermates

Studies were made of the distribution of gut hormones and neuropeptides in the gastrointestinal tract of mice with hereditary aganglionic colon (s1/ s1) and their normal littermates. Antisera to substance P, vasoactive intestinal polypeptide, and enkephalins demonstrated markedly diminished numbers of immunofluorescent nerve fibers in the aganglionic segment of colon; in contrast, in proximal colon and small intestine the distribution of peptidergic nerve fibers was essentially normal. Mucosql endocrine cells were demonstrated in the colon by antisera to substance P, somatostatin, glucagon, and cholecystokinin; in each case there were similar numbers of cells in s1/s1 and normal mice. Radioimmunoassays for vasoactive intestinal polypeptide and bombesin showed diminished concentrations of immunoreactive material in the aganglionic segment of colon of 16–17-day-old animals. However, assays for CCK—which is predominantly located in endocrine cells—showed similar concentrations of immunoreactive material throughtout the gut of s1/s1 and normal mice. It was of interest that the concentrations of immunoreactive substance P were lower than normal not only in the aganglionic segment of colon, but also in proximal colon and distal small intestine, and that at all ages the development of substance P in the intestine was delayed in the s1/s1 mice. The results are consistent with the idea that the constriction of the aganglionic segment of colon develops as a consequence of lack of intrinsic inhibitory neurons, possibly those containing vasoactive intestinal polypeptide. The presence of an aganglionic segment is attributable to delayed migration of the neuroblasts from the neural crest. Thus the finding of normal populations of gut endocrine cells in the aganglionic segment is further support for the view that the embryologic origin of gut endocrine cells is different to that of gut neurons.