An immunohistochemical study of the pancreatic endocrine cells of the Korean golden frog, Rana plancyi chosenica

The regional distribution and quantitative frequency of pancreatic endocrine cells were demonstrated in the Korean golden frog (Rana plancyi chosenica Okada), which is known as a Korean endemic species, for the first time, by immunohistochemical methods using specific mammalian antisera to insulin, glucagon, somatostatin and human pancreatic polypeptide (PP). In the pancreas of the Korean golden frog, all four endocrine cell types were demonstrated. Insulin- and glucagon-positive cells were located in the pancreas as single cells or islet-like clusters with frequencies of 85.90±18.28 and 54.30±8.77/1,000/1,000 cells, respectively. Somatostatin-containing cells were also dispersed in the pancreas as single cells or clusters but in the case of clusters, they are exclusively situated in the marginal regions of insulin- or glucagon-positive cell clusters. Cells stained for somatostatin cell frequency was 15.50±3.10/1000 cells. PP-containing cells were also distributed as single cells or clusters with frequency of 53.40±11.96/1,000 cells. Clusters consisted of PP-positive cells are distributed as a core type and a marginally distributed type. Overall, there were 40.84±3.81% insulin-, 26.02±1.71% glucagon-, 7.63±2.09% somatostatin- and 25.51±3.26% PP-IR cells.     

The distribution and frequency of endocrine cells in the splenic lobe of grass lizard (Takydromus wolteri). An immunohistochemical study

The regional distribution and frequency of the pancreatic endocrine cells in the splenic lobe of grass lizard, Takydromus wolteri, were studied by immunohistochemical (PAP) method using six types of specific mammalian antisera against bovine Sp-1/chromogranin (bCG), serotonin, insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). The pancreas was subdivided into two regions--islet kike and exocrine regions. The frequency of each immunoreactive (IR) endocrine cells was calculated as mean number/total 100 islet cells and as mean number/total 1,000 cells (including exocrine and endocrine cells) using automated image analysis process. In addition, the percentage of each IR cell was also calculated. All of six endocrine cells were demonstrated. They were dispersed in the whole pancreatic parenchyma between exocrine acinar cells, or they were also observed as islet like clusters. In islet-like regions, bCG-, insulin- and glucagon-IR cells were detected as one or two cell layer cords and they were located between this cell-cords with 14.30+/-5.62, 61.50+/-9.76 and 26.50+/-9.31/100 cells frequencies, respectively. However, somatostatin-IR cells were mainly located in the peripheral parts not in cell-cords with 12.40+/-4.86/100 cells, and no serotonin- and hPP-IR cells were demonstrated. In exocrine regions, all of bCG-, serotonin-, insulin-, glucagon-, somatostatin- and hPP-IR cells were detected and they occurred mainly among the exocrine parenchyma as solitary cells with 10.30+/-2.54, 0.80+/-0.63, 15.50+/-5.30, 5.80+/-2.66, 3.10+/-1.29 and 11.00+/-3.33/1000 cells frequencies, respectively. In addition, serotonin-IR cells were mainly located between epithelia and connective tissue of pancreatic duct. Overall, there were 0.58+/-0.49% serotonin-, 56.44+/-9.35% insulin-, 23.73+/-8.22% glucagon-, 11.28+/-3.03% somatostatin- and 7.97+/-2.02% hPP-IR cells.     

Distribution and frequency of endocrine cells in the pancreas of the ddY mouse: an immunohistochemical study

The regional distribution and frequency of pancreatic endocrine cells in ddY mice were studied by an immunohistochemical (peroxidase anti-peroxidase; PAP) method using four types of specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central portion. Most of glucagon- and somatostatin-IR cells were observed in peripheral regions although a somewhat smaller number of cells were also located in the central regions. HPP-IR cells were randomly distributed throughout the entire islets. In the exocrine pancreas, insulin-, glucagon-, somatostatin- and hPP-IR cells were detected; they occurred mainly among the exocrine parenchyma as solitary cells. Cell clusters consisted of only insulin- or only glucagon-IR cells and were distributed in the pancreas parenchyma as small islets. In addition, insulin- and glucagon-IR cells were also demonstrated in the pancreatic duct regions. Insulin-IR cells were located in the epithelium and sub-epithelial connective tissue regions as solitary cells and/or clusters (3-4 cells), and glucagon-IR cells were mainly located in the epithelium as solitary cells. Overall, there were 63.89+/-5.39% insulin-, 26.52+/-3.55% glucagon-, 7.25+/-2.83% somatostatin- and 1.90+/-0.58% hPP-IR cells. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells were found in the ddY mouse.     

An immunohistochemical study of the pancreatic endocrine cells of the nude mouse, Balb/c-nu/nu

The regional distribution and frequency of the pancreatic endocrine cells in the nude mouse, Balb/c-nu/nu were studied by immunohistochemical (peroxidase anti-peroxidase; PAP) methods using specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). The pancreas of the mouse was divided into two lobes, the splenic and duodenal lobes, and each lobe was subdivided into three regions, the pancreatic islets (central and peripheral regions), the exocrine region and the pancreatic duct region (consisting of duct epithelium and surrounding connective tissue--sub-epithelial connective tissue). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central region, and glucagon-, somatostatin and hPP-IR cells were located in the peripheral region regardless of the lobe. In the splenic part, glucagon-IR cells were also located in the central regions, and more numerous somatostatin-IR cells were detected in the central regions compared to those of the duodenal part. hPP-IR cells were restricted to the peripheral regions in both lobes but more numerous cells were detected in the duodenal portion as compared to those of the splenic portion. In the exocrine parenchyma of the splenic lobe, only insulin-, glucagon- and somatostatin-IR cells were detected.. Here, the insulin- and glucagon-IR cells formed cell clusters, while somatostatin-IR cells were present as solitary cells. In the exocrine region of the duodenal portion, only insulin-, somatostatin- and hPP-IR cells were observed, with the same distributional pattern as that found in the splenic lobe. However, clusters of cells consisting only of hPP-IR cells were distributed in the pancreas parenchyma as small islets. In the pancreatic duct region, only solitary hPP-IR cells were demonstrated in the sub-epithelial connective tissue regions of the splenic portion. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells, especially of the hPP-IR cells, were found in the nude mouse. In addition, somewhat different distributional patterns were found between the two pancreatic lobes.     

An immunohistochemical study of the pancreatic endocrine cells of the ddN mouse

The regional distribution and frequency of the pancreatic endocrine cells in the ddN mouse were studied using specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central region, and glucagon-, somatostatin and hPP-IR cells were located in the peripheral region regardless of the lobe. In the splenic part, glucagon-IR cells were also located in the central regions, and more numerous somatostatin-IR cells were detected in the central regions as compared with the duo-denal part. hPP-IR cells were restricted to the peripheral regions in both lobes but more numerous cells were detected in the duodenal portion. In the exocrine parenchyma of the splenic lobe, only insulin- and glucagon-IR cells were detected but all four kinds of IR cells were observed in the duodenal portion. In addition, insulin and hPP-IR cells were also demonstrated in the pancreatic duct regions. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells were found in the ddN mouse with somewhat different distributional patterns between the two pancreatic lobes.     

The endocrine pancreas of a squamate reptile, the desert lizard (Chalcides ocellatus). A histological and immunocytochemical investigation

The endocrine pancreas of the desert lizard (Chalcides ocellatus) was investigated histologically and immunocytochemically. The endocrine tissue was concentrated in the dorsal lobe, where it constituted about 7% of the total volume. In the ventral lobe the endocrine tissue formed approximately 1% of the total volume. Four endocrine cell types were observed in the pancreas of this species, namely insulin-, glucagon-, somatostatin- and pancreatic polypeptide (PP)-immunoreactive cells. The volume occupied by these cells was 1, 1, 0.6 and 0.3% of the total volume of the pancreas, respectively. Insulin-immunoreactive cells were located in the islet centre and comprised 3% of dorsal and 0.2% of the ventral lobe volume. Glucagon cells occurred at the islet periphery and amounted to 3 and 0.2% of the volume of the dorsal and ventral lobes, respectively. Somatostatin-immunoreactive cells were located at the islet periphery as well as in between the exocrine parenchyma. They constituted 1 and 0.2% of the volume of the dorsal and ventral lobes, respectively. PP-immunoreactive cells occurred mainly among the exocrine parenchyma as solitary cells. They formed only 0.03% of the volume of the dorsal lobe. The corresponding figure in the ventral lobe was 0.6%.     

The endocrine pancreas of a squamate reptile,the desert lizard (Chalcides ocellatus)

Summary The endocrine pancreas of the desert lizard (Chalcides ocellatus) was investigated histologically and immunocytochemically. The endocrine tissue was concentrated in the dorsal lobe, where it constituted about 7% of the total volume. In the ventral lobe the endocrine tissue formed approximately 1% of the total volume. Four endocrine cell types were observed in the pancreas of this species, namely insulin-, glucagon-, somatostain- and pancreatic polypeptide (PP)-immunoreactive cells. The volume occupied by these cells was 1, 1, 0.6 and 0.3% of the total volume of the pancreas, respectively. Insulin-immunoreactive cells were located in the islet centre and comprised 3% of dorsal and 0.2% of the ventral lobe volume. Glucagon cells occurred at the islet periphery and amounted to 3 and 0.2% of the volume of the dorsal and ventral lobes, respectively. Somatostatin-immunoreactive cells were located at the islet periphery as well as in between the exocrine parenchyma. They constituted 1 and 0.2% of the volume of the dorsal and ventral lobes, respectively. PP-immunoreactive cells occurred mainly among the exocrine parenchyma as solitary cells. They formed only 0.03% of the volume of the dorsal lobe. The corresponding figure in the ventral lobe was 0.6%.     

All-trans retinoic acid suppresses exocrine differentiation and branching morphogenesis in the embryonic pancreas

Recent evidence has shown that retinoic acid (RA) signalling is required for early pancreatic development in zebrafish and frog but its role in later development in mammals is less clear cut. In the present study, we determined the effects of RA on the differentiation of the mouse embryonic pancreas. Addition of all-trans retinoic acid (atRA) to embryonic pancreatic cultures induced a number of changes. Branching morphogenesis and exocrine differentiation were suppressed and there was premature formation of endocrine cell clusters (although the total area of beta cells was not different in control and atRA-treated buds). We investigated the mechanism of these changes and found that the premature formation of beta cells was associated with the early expression of high-level Pdx1 in the endocrine cell clusters. In contrast, the suppressive effect of RA on exocrine differentiation may be due to a combination of two mechanisms (i) up-regulation of the extracellular matrix component laminin and (ii) enhancement of apoptosis. We also demonstrate that addition of fibroblast growth factor (FGF)-10 is able to partially prevent apoptosis and rescue exocrine differentiation and branching morphogenesis in atRA-treated cultures but not in mice lacking the FGF receptor 2-IIIb, suggesting the effects of FGF-10 are mediated through this receptor.     

Dissociation of Langerhans islets in the rabbit after pancreatic duct ligation. Immunocytochemical and ultrastructural studies

In the rabbit, pancreatic duct ligation leads to serious disturbances of the pancreatic endocrine parenchyma. Immunocytochemical studies conducted over a short period (between 5 and 30 days post ligation) allow observation of a progressive dissociation of the Langerhans islets which initially affects the splenic part of the pancreas, a region where numerous large islets are found. This dissociation is followed by a dispersion of small heterologous endocrine cell clusters or isolated endocrine cells in a connective tissue which replace the exocrine parenchyma. On the 30th day after ligation ultrastructural studies show marked degranulation of the B cells demonstrating the great fragility of these cells. These observations of insular dissociation, scattering of the different endocrine cells and impairment of B cells are often reported in experimental and pathological studies of the pancreas.     

Amylin-like immunoreactivity in pancreatic X cells of the black-spotted frog Rana (Pelophylax) nigromaculata

In this study, we investigated the presence of ovoid or ellipsoidal amylin-immunoreactive cells of the pancreatic islets of the black-spotted frog Rana (Pelophylax) nigromaculata. Using double immunofluorescent staining, all amylin-immunoreactive cells were shown to be immuno-negative for insulin, glucagon, and somatostatin, and they were often observed in peripheral regions of clusters of insulin-immunoreactive cells. Under immunoelectron microscopy, amylin-immunoreactive signals were detected on the secretory granules in a specific type of endocrine cells. From our results, we conclude that the amylin-immunoreactive cells correspond to X cells among the 4 distinct types of endocrine cells (B, A/PP, D, and X) previously identified in the frog. Amylin secreted from X cells may regulate the hormone secretion from A/PP cells and/or B cells through a paracrine mechanism.     

Nestin-expressing cells in the pancreatic islets of Langerhans

The pancreatic islets of Langerhans produce several peptide hormones, predominantly the metabolically active hormones insulin and glucagon, which are critical for maintaining normal fuel homeostasis. Some evidence exists that pancreatic endocrine cells turn over at a slow rate and can regenerate in certain conditions. This could be due to the presence of pluripotent cells residing in the pancreas. Recently the intermediate filament protein nestin has been identified to be a marker for a multipotent stem cell in the central nervous system. Given the similarity between the pancreatic islets and neuronal cells, we hypothesized that stem cells expressing nestin might be present in the pancreas. Here we present evidence that a subset of cells in the pancreatic islets express the stem cell marker nestin. These cells might serve as precursors of differentiated pancreatic endocrine cells.     

Immunohistochemical study of the developing endocrine pancreas of the opossum (Didelphis virginiana)

Cells immunoreactive for insulin, glucagon, somatostatin, bovine pancreatic polypeptide and 5-hydroxytryptamine are found in the pancreas of the newborn opossum and of all later stages examined. All immunoreactive cell types are present in primary and secondary islets and within elements of the exocrine pancreas. Cells immunoreactive for glucagon, bovine pancreatic polypeptide, somatostatin and 5-hydroxytryptamine generally are confined to the periphery of secondary (intralobular) islets, whereas insulin-immunoreactive cells occupy the central region. Endocrine cells within primary (interlobular) islets are randomly scattered. A small number of pancreatic-polypeptide-immunoreactive cells are reactive for the amine 5-hydroxytryptamine also, but the reverse is not observed. The endocrine pancreas continues to differentiate and develop throughout postnatal life and into adulthood. Little difference was observed between the head and tail regions of the opossum pancreas for the measurements made.     

Immunohistochemical study of the distribution of endocrine cells in the gastrointestinal tract of the camel (Camelus bactrianus)

The regional distribution and relative frequency of endocrine cells in the gastrointestinal tract of the camel, Camelus bactrianus, were investigated using immunohistochemical methods. Ten types of immunoreactive (IR) endocrine cells were identified in this study. Among these cell types, only serotonin- and somatostatin-IR cells were detected in almost all regions of the gastrointestinal tract. Most of the cell types showed peak density in the pyloric gland region. The others showed restricted distribution: gastrin, cholecystokinin (CCK), motilin, bovine pancreatic polypeptide (BPP), and (gastric) substance P in the stomach; gastrin, CCK, BPP, gastric inhibitory polypeptide (GIP), glucagon, peptide tyrosine tyrosine (PYY) and substance P in the small intestine; and CCK, motilin, BPP, and PYY in the large intestine. Fundamentally the distribution pattern of endocrine cells in the gastrointestinal tract of the camel is similar to that of cattle. The distribution and frequency of endocrine cells in the glandular sac region are the same as those of the cardiac gland.     

The identification of the F-cell in the dog pancreas as the pancreatic polypeptide producing cell.

The endocrine cells of the processus uncinatus in the dog pancreas were investigated with special reference to the formerly known F-cell. The F-cell was detected frequently in the periphery of pancreatic islets as well as among exocrine tissue. In both localizations the F-cell shows similar ultrastructural features. Membrane-bound irregularly shaped secretory granules of variable electron density were seen. The cell possesses all features of an endocrine polypeptide secreting cell. Using the immunofluorescence and immunoperoxidase technique in the uncinate processus of the dog, we could reveal that the anti-sera against bovine pancreatic polypeptide (BPP) reacts with the cell which is localized at the same sites as the F-cell. We therefore conclude that the pancreatic F-cell is identical to the pancreatic polypeptide-producing cell. The other endocrine cell types of the dog pancreas are glucagon-producing A-cells, insulin-producing B-cells, and somatostatin-producing D-cells, as well as serotonin-producing EC-cells which are regularly present in the dog pancreatic islets and also scattered among exocrine tissue and the duct epithelial cells.     

An immunohistochemical study of pancreatic endocrine cells in SKH-1 hairless mice

The regional distribution and frequency of the pancreatic endocrine cells in the SKH-1 hairless mouse were studied by an immunohistochemical (peroxidase anti-peroxidase; PAP) method using four types of specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (PP). The pancreas of mice were divided into three portions; pancreatic islets, exocrine and pancreatic ducts. The pancreatic islets were further subdivided into three regions (central, mantle and peripheral region) according to their located types of immunoreactive cells. In the pancreatic islet portions, insulin-immunoreactive cells were located in the central and mantle regions with 84.60 +/- 7.65 and 33.00 +/- 12.45/100 cells frequencies, respectively, but most of somatostatin-, glucagon- and PP-immunoreactive cells were detected in the mantle and peripheral regions. In the mantle region, somatostatin-, glucagon- and PP-immunoreactive cells were demonstrated with 28.70 +/- 9.91, 52.00 +/- 14.05 and 2.60 +/- 1.51/100 cells frequencies, respectively, and showed 6.20 +/- 2.86, 15.30 +/- 5.31 and 21.50 +/- 10.28/100 cells frequencies, respectively in peripheral regions. However, glucagon-immunoreactive cells were also demonstrated in the central regions with 4.00 +/- 2.83/100 cells frequency. In the exocrine portions, insulin-, glucagon-, somatostatin- and PP-immunoreactive cells were demonstrated in the SKH-1 mouse with 0.90 +/- 0.74, 0.80 +/- 0.79,4.90 +/- 3.54 and 2.70 +/- 1.34/100 cells frequencies, respectively. In the pancreatic duct portions, insulin-, glucagon- and somatostatin-immunoreactive cells were demonstrated in the subepithelial connective tissues and showed islet-like appearances with 30.30 +/- 14.67, 2.70 +/- 3.13 and 5.90 +/- 4.23/100 cells frequencies, respectively. However, no PP-immunoreactive cells were demonstrated in these regions. In conclusion, some peculiar distributional patterns of pancreatic endocrine cells were found in the SKH-1 hairless mouse.     

The effect of diet on the Vervet monkey endocrine pancreas

Vervet monkeys (Cercopithecus aethiops) used for pancreatic endocrine cell distribution studies were found to have been maintained on different diets. Although the effect of dietary changes on the exocrine pancreas has been described in several animals, little, apart from the effect of malnutrition, has been reported for the endocrine pancreas. Reported here are pancreatic endocrine cell distributions in monkeys on a standard diet (n ? 3) compared with monkeys on an atherogenic diet (n = 3). Quantitation of immunolabelled pancreatic endocrine cell types revealed a significant 80% increase in A (glucagon) cell volume in monkeys on an atherogenic diet concomitant with a significant reduction in B (insulin) cell volume to approximately 60% of normal. This reflects a pattern of events that occurs in non-insulin dependent diabetes. An accompanying reduction in PP (pancreatic polypeptide) cell volumes supports our hypothesis that altering A and PP cell volumes could reflect differential gene expression in those cells in the adult in which glucagon and PP are co-localized.     

Pancreatic lineage analysis using a retroviral vector in embryonic mice demonstrates a common progenitor for endocrine and exocrine cells

The origin of pancreatic endocrine cells is unknown. Some studies have suggested that there is a common pancreatic progenitor which gives rise to both endocrine and exocrine cells, while others have suggested separate endocrine and exocrine lineages. Previous conclusions have been based on indirect data, such as the co-expression of molecular markers. We directly assessed the relationship between endocrine and exocrine cells during development using a lineage tracer. A replication-incompetent retrovirus was used to introduce the reporter gene alkaline phosphatase into single cells in explants of mouse embryonic pancreas. After a week in culture, the subsequent fate of the infected cells could then be determined. The results show that a common pancreatic progenitor cell exists, which gives rise to both endocrine and exocrine cells.     

Notch/Rbp-j signaling prevents premature endocrine and ductal cell differentiation in the pancreas

To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas.     

Cytochemistry and ultrastructure of frog pancreatic exo- and endocrine epithelium under normal conditions]

The pancreatic epithelium of intact frog has been studied using morphometry, cytochemistry and electron microscopy. A low level of DNA synthesis was shown to be characteristic of pancreatic epithelium of frogs caught in winter. The B/A cell volume ratio is 3.68 +/- 0.16 and 2.55 +/- 0.36 in small and median pancreatic islets, resp. A-, B-, D-cells are found in pancreatic islets, and intermediate acinar A and acinar B cells in intact frog pancreas.