Characterization of somatostatin receptors and associated signaling pathways in pancreas of R6/2 transgenic mice

The present study describes the status of somatostatin receptors (SSTRs) and their colocalization with insulin (β), glucagon (α) and somatostatin (δ) producing cells in the pancreatic islets of 11 weeks old R6/2 Huntington's Disease transgenic (HD tg) and age-matched wild type (wt) mice. We also determined expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and presynaptic marker synaptophysin (SYP) in addition to signal transduction pathways associated with diabetes. In R6/2 mice, islets are relatively smaller in size, exhibit enhanced expression and nuclear inclusion of mHtt along with the loss of insulin, glucagon and somatostatin expression. In comparison to wt, R6/2 mice display enhanced mRNA for all SSTRs except SSTR2. In the pancreatic lysate, SSTR1, 4 and 5 immunoreactivity decreases whereas SSTR3 immunoreactivity increases with no discernible changes in SSTR2 immunoreactivity. Furthermore, at the cellular level, R6/2 mice exhibit a receptor specific distributional pattern of SSTRs like immunoreactivity and colocalization with β, α and δ cells. While GAD expression is increased, TH and SYP immunoreactivity was decreased in R6/2 mice, anticipating a cross-talk between the CNS and pancreas in diabetes pathophysiology. We also dissected out the changes in signaling pathway and found decreased activation and expression of PKA, AKT, ERK1/2 and STAT3 in R6/2 mice pancreas. These findings suggest that the impaired organization of SSTRs within islets may lead to perturbed hormonal regulation and signaling. These interconnected complex events might shed new light on the pathogenesis of diabetes in neurodegenerative diseases and the role of SSTRs in potential therapeutic intervention.     

饥饿状态大鼠胰腺胰高血糖素和胰岛素变化的定量分析

用免疫组织化学方法结合图象分析技术对饥饿状态大鼠胰岛Ａ、Ｂ细胞中胰高血糖素（Ｇｌｕｃａｇｏｎ,Ｇｌｕ）和胰岛素（Ｉｎｓｕｌｉｎ,Ｉｎｓ）的免疫反应强度进行定量分析。结果表明：与正常对照相比,饥饿大鼠胰岛Ａ细胞中的Ｇｌｕ含量明显下降,Ｂ细胞中Ｉｎｓ含量明显升高。提示饥饿可导致Ｇｌｕ释放增加,Ｉｎｓ释放减少。与饥饿５天大鼠组相比较,饥饿５天后静脉注射葡萄糖组９０ｍｉｎ后胰岛内Ｇｌｕ含量明显升高,Ｉｎｓ含量无显著变化。提示：静脉注射葡萄糖可快速作用于胰岛Ａ细胞,减少Ｇｌｕ释放,但其对Ｂ细胞作用缓慢。从而为进一步阐明葡萄糖对胰岛Ａ、Ｂ细胞的不同作用机制提供形态学依据。     

饥饿状态大鼠胰腺高血糖素和胰岛素变化的定量分析

用免疫组织化学方法结合图象分析技术对饥饿状态大鼠胰岛Ａ、Ｂ细胞中胰因糖素和胰岛素的免疫反应强度进行定量分析。结果表明：与正常对照相比,饥饿大鼠胰岛细胞中的Ｇｌｕ含量明显下降,Ｂ细胞中Ｉｎｓ含量明显升高。提示饥饿可导致Ｇｌｕ释放增加,Ｉｎｓ和减少。与饥饿５天大鼠线要比较,饥饿５天后静脉注射葡萄糖组９０ｍｉｎ后胰岛内Ｇｌｕ含量明显升高,Ｉｎｓ含量无显著变化。提示：静脉注射葡萄糖要快速作用下胰岛Ａ细胞,     

Development of hormonal peptides and processing enzymes in the embryonic avian pancreas with special reference to co-localisation

Studies on the developing mammalian pancreas have suggested that insulin and glucagon co-exist in a transient cell population and that peptide YY (PYY) marks the earliest developing endocrine cells. We have investigated this in the embryonic avian pancreas, which is characterised by anatomical separation of insulin and glucagon islets. Moreover, we have compared the development of the endocrine cells to that of processing enzymes involved in pancreatic hormone biosynthesis. PYY-like immunoreactivity occurred in islet cells from the youngest stages examined: it increased in amount from approximately 5 days of incubation and was co-localised with glucagon and to a lesser extent with insulin. Insulin and glucagon cells were numerous: co-existence of the two peptides in the same cells was but rarely observed. From the youngest stages examined, prohormone convertase (PC) 1/3-like immunoreactivity was detected in insulin cells and PC2-, 7B2- and carboxypeptidase E-like immunoreactivity in both glucagon and insulin cells. We conclude that: (1) PYY-like immunoreactivity occurs in avian islet cells but generally in lesser amounts than in mammals at the earlier stages, (2) the paucity of cells co-expressing insulin and glucagon indicate that all avian insulin cells do not pass through a stage where they co-express glucagon and (3) the early expression of the enzymes responsible for the processing of prohormones suggests that this process is initiated soon after islet cells first differentiate.     

Factors influencing insulin and glucagon secretion in lean and genetically obese mice.

The control of insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.     

Expression of the protein tyrosine phosphatase-like protein IA-2 during pancreatic islet development.

A tyrosine phosphatase-like protein, IA-2, is a major autoantigen in Type 1 diabetes but its role in islet function is unclear. Tyrosine phosphorylation mediates regulation of cellular processes such as exocytosis, cell growth, and cell differentiation. To investigate the potential involvement of IA-2 in islet differentiation and insulin secretion, we analyzed by immunohistochemistry expression of IA-2 during islet development in fetal rats and during the maturation of insulin secretory responses after birth. In the fetus, IA-2 immunoreactivity was detected in primitive islets positive for insulin and glucagon at 12 days' gestation. Subsequently, IA-2 was only weakly detectable in the fetal pancreas. In neonatal rat, a progressive increase in IA-2 immunoreactivity was observed in islets from very low levels at 1 day of age to moderate labeling at 10 days. In the adult, relatively high levels of IA-2 were detected in islets, with heterogeneous expression in individual cells within each islet. IA-2 marks a population of endocrine cells that transiently appear early in pancreatic ontogeny. Islet IA-2 expression reappears after birth concomitant with the development of mature insulin secretory responses, consistent with a role for this protein in regulated hormone secretion.     

Cell lineage analysis of pancreatic islet development: glucagon and insulin cells arise from catecholaminergic precursors present in the pancreatic duct

We have previously reported that cells transiently expressing tyrosine hydroxylase (TH), the first enzyme of the catecholamine biosynthetic pathway, are present in the pancreas of mouse embryos from prenatal Day 11 (E11) and that, at E12, some TH cells contain glucagon. Cells containing TH were also found in adults which, unlike the TH cells of embryos, did not contain glucagon (G. Teitelman, T. H. Joh, and D. J. Reis (1981). Proc. Natl. Acad. Sci. 78, 5225). These findings suggested to us that the TH cells of embryonic pancreas were the precursors of glucagon cells of adults. In this study we used immunocytochemical and autoradiographic techniques to determine whether cells containing TH (a) were present in pancreas throughout pre- and postnatal development, (b) were localized to a specific region of the gland, (c) contained insulin at any time, and (d) proliferated. We found that TH cells were present in pancreas throughout life. In embryos, cells containing TH localized only along the pancreatic duct, also contained either glucagon or insulin, and were able to proliferate. In contrast, after birth, the pancreatic duct contained no TH cells. Cells containing TH in postnatal and adult mice also differed from embryonic TH cells in that they were found in all islets, contained insulin but not glucagon, and did not synthesize DNA, and hence did not proliferate. These findings suggest that progenitor cells that contain catecholamines and are present in the pancreatic duct give rise to glucagon and insulin cells of adult islets. They also indicate that the TH-insulin cells of postnatal and adult mice are not stem cells but are postmitotic cells that appear in the islets after birth.     

Nesfatin-1 stimulates glucagon and insulin secretion and beta cell NUCB2 is reduced in human type 2 diabetic subjects

Nesfatin-1 is a novel anorexigenic regulatory peptide. The peptide is the N-terminal part of nucleobindin 2 (NUCB2) and is expressed in brain areas regulating feeding. Outside the brain, nesfatin-1 expression has been reported in adipocytes, gastric endocrine cells and islet cells. We studied NUCB2 expression in human and rodent islets using immunocytochemistry, in situ hybridization and western blot. Furthermore, we investigated the potential influence of nesfatin-1 on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-1 (832/13) cells. The impact of type 2 diabetes (T2D) and glucolipotoxicity on NUCB2 gene expression in human islets and its relationship to insulin secretory capacity and islet gene expression was studied using microarray. Nesfatin-1 immunoreactivity (IR) was abundant in human and rodent beta cells but absent in alpha, delta, PP and ghrelin cells. Importantly, in situ hybridization showed that NUCB2 mRNA is expressed in human and rat islets. Western blot analysis showed that nesfatin-1 IR represented full length NUCB2 in rodent islets. Human islet NUCB2 mRNA was reduced in T2D subjects but upregulated after culture in glucolipotoxic conditions. Furthermore, a positive correlation between NUCB2 and glucagon and insulin gene expression, as well as insulin secretory capacity, was evident. Nesfatin-1 enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-1 (832/13) cells. On the other hand, nesfatin-1 caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that nesfatin-1 is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.     

Glucagon- and glicentin-immunoreactive cells in the human digestive tract

Summary The distribution and cellular location of substances reacting with anti-glucagon or anti-glicentin sera, i.e., glucagon-like and glicentin-like immunoreactivities, were studied in the human digestive tract using the immunofluorescence and immunoperoxidase methods. Both types of immunoreactivity were (1) absent in the antrum, (2) abundant in cells located at the periphery of pancreatic islets, (3) unevenly present in cells scattered in the epithelium of the small intestinal mucosa, the glicentin-immunoreactive cells being particularly abundant in the ileum. In the pancreas, and, when simultaneously present, in the intestine, both glucagon and glicentin immunoreactivities were located in the same cells.The precise ultrastructural location of each immunoreactivity was readily made using colloidal gold and ferritin tracers on ultrathin sections of glutaraldehyde-osmium fixed and epoxy resin-embedded tissues. In the pancreas, both glucagon and glicentin immunoreactivities were found in the granules of the A-type cells; the glucagon immunoreactivity was only present in the core of the granule, whereas the glicentin immunoreactivity was found either in the peripheral halo only, or throughout the entire granule. In the small intestine, both immunoreactivities were located inside the granules of the L-type cells.Quantitative specificity tests suggested that the glucagon- and the glicentin-like substances of the pancreas differ from those found in the intestine.Work supported by INSERM, A.T.P. number: 167539     

Immunocytochemical evidence for the presence of S-100 protein in insulin-containing cells of cultured fetal rat islets

S-100 protein was long considered to be specific to glial and Schwann cells, but was subsequently proved to be present in various organs. In particular, S-100 proteinimmunoreactivity was demonstrated in the parathyroid gland, adenohypophysis and endocrine pancreas. In the present study cultured fetal rat islets were investigated in view of the possible presence of S-100 protein immunoreactivity in their cells. In the initial 5-day period, continuity between islets and ducts could be demonstrated, and the islets appeared to bud from the ducts. During this time, S-100 protein-immunoreactive cells were found in either the budding islets or ducts. The colocalization of S-100 protein and insulin was demonstrated immunocytochemically. In contrast, the newly formed islets from endocrine monolayers did not display S-100 protein immunoreactivity. After this initial period, numerous free-floating islets were observed, but only some of them contained S-100 protein immunoreactivity. S-100 protein-immunoreactive cells had the same distribution as those storing insulin, again suggesting the coexistence of the two peptides. The results suggest that S-100 protein might be involved in the regulation of islet function.     

The role of leucine-enkephalin on insulin and glucagon secretion from pancreatic tissue fragments of normal and diabetic rats

Leucine-enkephalin (Leu-Enk) has been shown to be present in endocrine cells of the rat pancreas and may play a role in the modulation of hormone secretion from the islets of Langerhans. Since little is known about the effect of Leu-Enk on insulin and glucagon secretion, it was the aim of this study to determine the role of Leu-Enk on insulin and glucagon secretion from the isolated pancreatic tissue fragments of normal and diabetic rats. Pancreatic tissue fragments of normal and streptozotocin-induced diabetic rats were incubated for 1 h with different concentrations of Leu-Enk (10(-12)-10(-6)M) alone or in combination with either atropine or yohimbine or naloxone. After the incubation period the supernatant was assayed for insulin and glucagon using radioimmunoassay techniques. Leu-Enk (10(-12 )-10(-6)M) evoked large and significant increases in insulin secretion from the pancreas of normal rats. This Leu-Enk-evoked insulin release was significantly (p < 0.05) blocked by atropine, naloxone and yohimbine (all at 10(-6)M). In the same way, Leu-Enk at concentrations of 10(-12)M and 10(-9)M induced significant (p < 0.05) increases in glucagon release from the pancreas of normal rats. Atropine, yohimbine but not naloxone significantly (p < 0.05) inhibited Leu-Enk-evoked glucagon release from normal rat pancreas. In contrast, Leu-Enk failed to significantly stimulate insulin and glucagon secretion from the pancreas of diabetic rats. In conclusion, Leu-Enk stimulates insulin and glucagon secretion from the pancreas of normal rat through the cholinergic, alpha-2 adrenergic and opioid receptor pathways.     

Development of the islets, exocrine pancreas, and related ducts in the Nile tilapia, Oreochromis niloticus (Pisces: Cichlidae)

Pancreatic development and the relationship of the islets with the pancreatic, hepatic, and bile ducts were studied in the Nile tilapia, Oreochromis niloticus, from hatching to the onset of maturity at 7 months. The number of islets formed during development was counted, using either serial sections or dithizone staining of isolated islets. There was a general increase in islet number with both age and size. Tilapia housed in individual tanks grew more quickly and had more islets than siblings of the same age left in crowded conditions. The pancreas is a compact organ in early development, and at 1 day posthatch (dph) a single principal islet, positive for all hormones tested (insulin, SST-14, SST-28, glucagon, and PYY), is partially surrounded by exocrine pancreas. However, the exocrine pancreas becomes more disseminated in older fish, following blood vessels along the mesenteries and entering the liver to form a hepatopancreas. The epithelium of the pancreatic duct system from the intercalated ducts to the main duct entering the duodenum was positive for glucagon and SST-14 in 8 and 16 dph tilapia. Individual insulin-immunopositive cells were found in one specimen. At this early stage in development, therefore, the pancreatic duct epithelial cells appear to be pluripotent and may give rise to the small islets found near the pancreatic ducts in 16-37 dph tilapia. Glucagon, SST-14, and some PPY-positive enteroendocrine cells were present in the intestine of the 8 dph larva and in the first part of the intestine of the 16 dph juvenile. Glucagon and SST-14-positive inclusions were found in the apical cytoplasm of the mid-gut epithelium of the 16 dph tilapia. These hormones may have been absorbed from the gut lumen, since they are produced in both the pancreatic ducts and the enteroendocrine cells. At least three hepatic ducts join the cystic duct to form the bile duct, which runs alongside the pancreatic duct to the duodenum.     

Porcine islet isolation, cellular composition and secretory response

Porcine islets were isolated by infusion of a warm collagenase solution into whole pancreata followed by static incubation at 37 degrees C for 15 minutes. The pancreata were then chopped into small pieces and the free islets purified by filtration and centrifugation over a ficoll gradient. The insulin:amylase ratio of the islets compared to that in the intact pancreas was determined in 19 pancreata and indicates that the isolated islets were of a high degree of purity. The distribution of insulin, glucagon, somatostatin and pancreatic polypeptide containing cells in pig pancreas sections was compared with that in rat. Porcine islets were much smaller and less well defined than rat islets with infiltration of acinar material even into the islet core. The levels of insulin, glucagon and somatostatin in porcine pancreas and isolated porcine islets were measured using conventional radioimmunoassay techniques. The ratio of these hormones in the pancreas was 105.1:5.8:1 respectively, and in the islets 105.1:0.68:0.087 respectively. Fragmentation of the islets during the isolation may have led to the loss of glucagon and somatostatin-containing cells. Islets cultured overnight and tested with a range of glucose concentrations for one hour did not show a significant stimulation of insulin secretion in the presence of 8.3 mM or 16.7 mM glucose compared to that in 2.8 mM glucose. However freshly isolated islets challenged with 8.3 mM, 13.9 mM and 22.2 mM glucose showed a 1.8 fold, 2.0 fold and 2.3 fold response respectively, over that in 2.8 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

An immunocytochemical study of endocrine pancreas of snakes

Summary The pancreas from eleven species of snakes representing both advanced and primitive families has been investigated for the presence of eleven regulatory peptides reported to occur in the mammalian endocrine pancreas. Of the eleven peptides studied, insulin, pancreatic glucagon and somatostatin were present in endocrine cells within the islets of all the species investigated. The neuropeptide, vasoactive intestinal polypeptide, was located within nerve terminals innervating the islets in the Boidinae, Colubrinae, Elaphidae and Crotalidae but absent from the Natricinae investigated.No immunoreactivity was demonstrable with the antisera to substance P, met-enkephalin, C-terminal gastrin, bombesin, glicentin and gastric inhibitory polypeptide. Pancreatic polypeptide-like immunoreactivity was demonstrable only in the boid snakes and exclusively stained by a C-terminal specific antiserum.     

Silymarin induces recovery of pancreatic function after alloxan damage in rats

Alloxan has been widely used to produce experimental diabetes mellitus syndrome. This compound causes necrosis of pancreatic beta-cells and, as is well known, induces oxidant free radicals which play a relevant role in the etiology and pathogenesis of both experimental and human diabetes mellitus. Previously we have reported hypoglycemic and antilipoperoxidative actions of silymarin in serum and pancreatic tissue respectively. The aim of this study was to test whether silymarin could reduce the hyperglycemia and revert the pancreatic damage in alloxan treated rats, tested with silymarin in two protocols: using both compounds simultaneously for four or eight doses, or using the compound 20 days after alloxan administration for 9 weeks. Serum glucose and insulin were determined, and pancreatic fragments were used for histology and insulin immunohistochemistry. Pancreatic islets were isolated to assess insulin and Pdx1 mRNA expression by RT-PCR. Our results showed that 72 hours after alloxan administration, serum glucose increased and serum insulin decreased significantly, whereas pancreatic tissue presented morphological abnormalities such as islet shrinkage, necrotic areas, loss of cell organization, widespread lipoid deposits throughout the exocrine tissue, and loss of beta cells, but insulin and glucagon immunoreactivity was scattered if any. In contrast the pancreatic tissue and both insulin and glucose serum levels of rats treated with silymarin were similar to those of control animals. In addition, insulin and glucagon immunoreactive cells patterns in Langerhans islets were also normal, and normal insulin and Pdx1 mRNA expression patterns were detected during pancreatic recovery in Langerhans islets. The overall results suggest that silymarin induces pancreatic function recovery demonstrated by insulin and glucagon expression protein and normoglycemia after alloxan pancreatic damage in rats.     

A comparative study of the portal vessels connecting the endocrine and exocrine pancreas, with a discussion of some functional implications.

We have examined the pattern of the capillaries of the pancreas in rabbits, rats, mice, guinea-pigs, cats and baboons, using arterial and venous injections of Berlin blue. In all these species we found extensive, direct connexions between the capillary beds of the islets and the exocrine tissue of the gland, forming a highly developed portal system. Some of the functional implications of these vascular connexions are discussed, particularly the influence of the islet hormones insulin, glucagon and somatostatin upon the exocrine cells.     

Somatostatin-induced inhibition of insulin secretion from isolated islets of rat pancreas in presence of glucagon.

In order to study the oeffect of somatostatin on the endocrine pancreas directly, islets isolated from rat pancreas by collagenase were incubated for 2 hrs 1) at 50 and 200 mg/100 ml glucose in the absence and presence of somatostatin (1, 10 and 100 mg/ml) and2) at 200 mg/100 ml glucose together with glucagon (5 mug/ml), with or without somatostatin (100 ng/ml). Immunologically measurable insulin was determined in the incubation media at 0, 1 and 2 hrs. Insulin release was not statistically affected by any concentration stomatostatin. On the other hand, somatostatin exerted a significant inhibitory action on glucagon-potentiated insulin secretion (mean +/- SEM, mu1/2 hrs/10 islets: glucose and glucagon: 1253 +/- 92; glucose, glucagon and somatostatin: 786 +/- 76). The insulin output in th epresence of glucose, glucagon and somatostatin was also significantly smaller than in thepresence of glucose alone (1104 +/- 126) or of glucose and somatostatin (1061 +/- 122). The failure of somatostatin to affect glucose-stimulated release of insulin from isolated islets contrasts its inhibitory action on insulin secretion as observed in the isolated perfused pancreas and in vivo. This discrepancy might be ascribed to the isolation procedure using collagenase. However, somatostatin inhibited glucagon-potentiated insulin secretion in isolated islets which resulted in even lower insulin levels than obtained in the parallel experiments without glucagon. It is concluded that the hormone of the alpha cells, or the cyclic AMP system, might play a part in the machanism of somatostatin-induced inhibition of insulin release from the beta-cell.