Neonatal Rat Pancreas Research Articles

Localization of microRNA-375 in perinatal rat pancreas

Abstract MicroRNA (miRNA)-375 is highly expressed in the pancreatic endocrine islets. Maintaining appropriate miR-375 levels is very important for beta cell development, function and proliferation. The aim of the current study was to investigate the regulation and localization of miR-375 in rat perinatal pancreas at embryonic day 20 (E20), postnatal day 0 (D0) and day 2 (D2). Expression levels of miR-375 were measured by in situ hybridization on fixed neonatal rat pancreas. Interestingly, while miR-375 was detectable at robust levels at all three time points, the major site of expression of miR-375 at D0 and D2 was in pancreatic exocrine cells. Our data show that miR-375 has a dynamic change of expression in pancreatic exocrine tissue during the perinatal period. Moreover, these findings indicate that pancreatic endocrine cells may not always be the major source of expression of miR-375 in pancreas. We suggest that the marked change of miR-375 levels in exocrine cells following birth could regulate processes involved in the adaptation of the exocrine pancreas to digestion of external nutrients derived from milk.

Investigating the Histological effects of industrial and oil pollutant dust in the air of Assaluyeh, on the pancreas and ovary

Today air pollution as a very important issue nationally and internationally known. The annual air pollution leads to disease and death of thousands of people around the world. Although some countries take measures in order to minimize the amount of environmental pollutants, but there are still an increase in morbidity and mortality caused by air pollution. Scientific findings have shown a wide range of air pollution effects on the. Air pollution can have devastating effects on children's health, this article tries to explore the effects of oil contamination and pollutants in the air of Assaluyeh on pancreas and ovary of maternal and neonatal rats.

Mesothelin promotes cell proliferation in the remodeling of neonatal rat pancreas.

To investigate the effect of mesothelin in the remodeling of the endocrine pancreas in neonatal rats. Overexpression or downregulation of mesothelin expression in INS-1 cells was carried out to investigate the effect of mesothelin during cell proliferation and cell apoptosis in vitro. Adenovirus-mediated RNA interference was performed to block mesothelin in vivo to directly assess the role of mesothelin in the remodeling of the endocrine pancreas in neonatal rats. Exogenous overexpression of mesothelin promoted cell proliferation, cell colony formation and enhanced cell resistance to apoptosis of INS-1 cells. Down-regulation of mesothelin made no difference in cell proliferation and apoptosis compared with that in the control group. After an injection of adenovirus-mesothelin, a significantly increased number of small islets appeared, and the expression of PCNA was decreased on day 7 and day 14 compared with the Ad-EGFP group. Mesothelin was able to promote β cell proliferation in the remodeling stage of neonatal rats. Mesothelin may have an important role in the remodeling of the endocrine pancreas in neonatal rats.

Streptozotocin-induced expression of Ngn3 and Pax4 in neonatal rat pancreatic α-cells.

To investigate the mechanism behind β-cell regeneration in neonatal rat pancreas treated with streptozotocin (STZ). Neonatal Sprague Dawley rats were intraperitoneally injected with 70 mg/kg STZ. Body weight, pancreas weight and blood glucose were recorded every two days after the treatment. To identify the expression and location of transcription factors in the rat pancreas, double immunofluorescent staining was performed using antibodies to specific cell markers and transcription factors. Expression of Neurogenin 3 (Ngn3), a marker for endocrine precursor cells, was observed by immunofluorescence in a few β-cells and many α-cells. The expression reached a peak 12 d after treatment. Pax4, a transcription factor that lies downstream of Ngn3 and plays an important role in β-cell differentiation, was also expressed in the α-cells of STZ-treated rats. We did not observe significant changes in Nkx6.1, which is essential for β-cell maturation in the treated rats. α-cells dedifferentiated into endocrine precursor cells and acquired the ability to dedifferentiate in the neonatal rat pancreas after STZ treatment.

A follow-up study of cell-mediated cytotoxicity and beta cell function in type I diabetes.

In 20 patients with a newly diagnosed type I diabetes mellitus a cytotoxic effect of blood lymphocytes against beta cells of the pancreas of neonatal rats could be demonstrated. This effect remained nearly unchanged during the first 12 months of control. During the course up to 18 months, the cytotoxicity decreased significantly. After stimulation with glucose and glucagon, a C-peptide secretion was demonstrated in all patients during the first 12 months but it decreased thereafter. The follow-up study showed cell-mediated immune reactions against beta cells in type I diabetics as long as the existence of beta cells can be assumed on the basis of functional tests. Thus the immune process seems to depend on the presence of the specific antigen.

Identification and action of N-myc downstream regulated gene 4 A2 in rat pancreas

Pancreatic islets and acinar tissue develop from duct epithelium and share expression of several transcription factors and other molecular markers also involved with the development of neural tissues. We examined rat pancreatic tissue from fetal life until adulthood for the expression of N-myc downstream regulated gene 4 (Ndrg4), a gene shown to be expressed during neuronal cell differentiation. Isolated pancreatic ducts from neonatal rats were maintained in culture and gave rise to clusters of cells expressing nestin (NES) and PDX-1, which subsequently contained immunoreactive glucagon. Using reverse transcription PCR (RT-PCR), we identified mRNA expression and immunoreactive protein presence for NDRG4 in cultured duct-derived cells, and brain of neonatal rats. By PCR cloning of the ductal cell-derived DNA the molecular form of NDRG4 expressed in pancreatic ducts and ARIP rat pancreatic cells was identified as NDRG4A2, and its presence in intact pancreas of fetal and neonatal rats was demonstrated by immunohistochemistry. Incubation of ARIP cells with glucagon-like polypeptide-1 (GLP-1), increased the expression of NDRG4A2 and PDX-1, while decreasing DNA synthesis and promoting the appearance of glucagon-positive cells. This inhibitory effect of GLP-1 on DNA synthesis and the stimulatory effect on endocrine differentiation were reversed when the translation of NDRG4A2 was prevented using siRNA. These findings indicate that NDRG4A2 is expressed in pancreatic duct cells under GLP-1 control and may be related to a reduction in proliferation and the onset of the pancreas cell differentiation.

Expression of ghrelin and calcium channels in pancreas of neonatal rats with different birth weight

To investigate the effect of the intrauterine environment on pancreas ghrelin and L-type calcium channels of islet like cell clusters (ICCs) in neonatal rats. Different birth weight neonatal rat models were established. The samples of entire pancreas or ICCs were collected at the first day after birth. Ghrelin, Cav1.2, Cav1.3 mRNAs and proteins were determined using real-time RT-PCR and immunohistochemistry respectively. The real-time RT-PCR revealed that ghrelin mRNA in SGA group were significantly higher than that of the AGA group (P <0.05). Cav1.2 mRNA of SGA group and LGA group was significantly lower than that of the AGA group; Cav1.3 mRNA of SGA group was significantly lower than that of the AGA group (P <0.05). The results of integral OD value of immunohistochemistry were consistent with those of real-time RT-PCR. Intrauterine nutritional status may affect pancreatic endocrine cells differentiation and maturity, resulting in the difference of expression ghrelin and calcium channels in ICCs.

Ontogeny of Regeneration of β-Cells in the Neonatal Rat after Treatment with Streptozotocin

We induced partial beta-cell loss within the pancreas of neonatal rats using streptozotocin (STZ) to better characterize the mechanisms leading to beta-cell regeneration postnatally. Rats were administered either STZ (70 mg/kg) or buffer alone on postnatal d 4, and the endocrine pancreas was examined between 4 and 40 d later. STZ-treated rats showed an approximately 60% loss of existing beta-cells and a moderate hyperglycemia (<15 mm glucose), with levels returning to near-control values after 20 d. Within preexisting islets, there was increased cell proliferation in both insulin- and glucagon-positive cells at 8 d as well as alpha-cell hyperplasia. These were associated with increased pancreatic content and circulating levels of glucagon. Pancreatic levels of glucagon-like polypeptide-1 (GLP-1) were increased 8 d after STZ compared with control values, and the GLP-1/glucagon ratio changed in favor of GLP-1. Administration of a GLP-1 receptor antagonist, GLP-1-(9-39), resulted in decreased recovery of beta-cells after STZ and worse glucose tolerance. Atypical glucagon-positive cells were found within islets that colocalized pancreatic duodenal homeobox-1 or glucose transporter-2. Pancreatic levels of insulin mRNA did not return to control values until 40 d after STZ. Insulin-positive cells were found after 8 d that colocalized glucagon and GLP-1. The model shows that the pancreas of the young rat can rapidly regenerate a loss of beta-cells, and this is associated with hyperplasia of alpha-cells with an altered phenotype of increased GLP-1 synthesis. The target cells of GLP-1 probably include immature beta-cells that coexpress proglucagon.

Promotion of beta-cell differentiation by conophylline in fetal and neonatal rat pancreas.

Conophylline is a vinca alkaloid extracted from the tropical plant Ervatamia microphylla and has been shown to induce differentiation of pancreatic AR42J cells. In the present study, we investigated the effect of conophylline on the differentiation of pancreatic precursor cells. In the rat pancreatic rudiment in organ culture, conophylline inhibited the formation of cystic structure and increased the number of insulin-positive cells. Conophylline also markedly increased the expression of mRNA for insulin and the number of pancreatic duodenal homeobox-1-positive cells. These effects of conophylline were similar to those of activin A. We also examined the effect of conophylline on neonatal rats treated with streptozotocin, a model of type 2 diabetes. Treatment with conophylline significantly reduced the plasma glucose concentration and improved glucose tolerance in response to glucose loading. The insulin content and the beta-cell mass at 2 months were significantly increased by conophylline. The number of islet-like cell clusters and pancreatic duodenal homeobox-1-positive ductal cells was greater in conophylline-treated rats. These results suggest that conophylline induces differentiation of pancreatic precursor cells and increases the formation of beta-cells.

Immunocytochemical detection of ornithine decarboxylase.

Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouin's fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats. (J Histochem Cytochem 47:1395-1404, 1999)

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine.

We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo) and 200 microM carbamylcholine (Cch). No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively). High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.

Gastrin effects on growth and exocrine secretion in the neonatal rat pancreas.

In adult rats, cholecystokinin (CCK) binds to pancreatic CCK-A receptors and stimulates exocrine secretion and pancreatic growth. In newborn rats there is very little mature intestinal CCK and few pancreatic CCK-A receptors. However, there is an abundant source of the related peptide gastrin, and in addition, there are pancreatic CCK-B/gastrin receptors. In this study, we have explored the hypothesis that gastrin may "deputise" for CCK in the neonate and cause pancreatic exocrine secretion, growth, and maturation. Newborn rats were injected intraperitoneally with the gastrin antagonist, C1-988, or placebo for 5 days and sacrificed on day 6. Body weight (CI-988, 8.25 +/- 0.48 g; placebo, 10.17 +/- 0.93 g) and pancreatic weight (19.41 +/- 1.5 vs. 21.58 +/- 1.72 mg) were the same. Protein (1,202 +/- 78 vs. 1,254 +/- 56 micrograms), amylase (22.3 +/- 0.9 vs. 19.2 +/- 1.00 U), RNA (117 +/- 7 vs. 126 +/- 5 micrograms), and DNA (95 +/- 8 vs. 100 +/- 7 micrograms) contents of the pancreas were similar in the two groups. The weight of the stomach was reduced (p = 0.04) in the treated group (50.8 +/- 2.5 mg) compared to the placebo group (59.8 +/- 3.7 mg). In both neonates and adults, CCK-8 was approximately 1,000 times more potent than gastrin-17 in stimulating amylase release from isolated pancreatic acini. This suggests that enzyme release is probably mediated by the same receptors in both adults and neonates, and in adults previous work has shown this to be the CCK-A receptor. In conclusion, these data show that gastrin has very weak effects on neonatal pancreatic exocrine secretion, and furthermore, gastrin is not essential for growth and maturation of the neonatal pancreas. The role of gastrin and CCK-B/gastrin receptors in the neonatal pancreas remains speculative.

Preprotachykinin-A gene expression occurs transiently in the developing rat endocrine pancreas and can be regulated in RINm5F cells.

We have reported previously that the rat insulinoma cell lines, RINm5F and RINr1046-38, express the preprotachykinin(PPT)-A gene, which encodes the tachykinin peptides, substance P and neurokinin A. Because endocrine cells of the adult rat pancreas do not appear to express PPT-A, we investigated whether the gene is expressed by rat pancreatic endocrine cells during development. We used immunohistochemistry, employing different substance P and neurokinin A antibodies, to show that many endocrine cells of the fetal and neonatal rat pancreas synthesise these products of PPT-A gene expression. Colabeling experiments revealed that a significant number of both insulin-containing and non-insulin-containing cells express tachykinins. After postnatal day 20, the number of tachykinin-immunoreactive pancreatic islet cells declines and, as already reported, none were detected in the adult rat pancreas. The transient expression of PPT-A by the developing endocrine pancreas is a novel finding. Substance P and neurokinin A are known to have trophic actions and may serve as growth factors during pancreatic islet development. PPT-A gene expression by RINm5F and RINr1046-38 cells is further evidence that these cells resemble developing pancreatic endocrine cells. They are potentially valuable as unique models for studying the regulation of tachykinin biosynthesis. We provide evidence in this study, using quantitative PPT-A messenger RNA analysis, that PPT-A expression in RINm5F cells may be up-regulated by activation of protein kinase C, down-regulated by activation of glucocorticoid receptors, and is not significantly affected by changes in intracellular cAMP levels.

Evidence for the role of pancreatic acinar cells in the production of ornithine and guanidinoacetic acid by L-arginine:glycine amidinotransferase.

L-Arginine:glycine amidinotransferase (transamidinase) occurs at high concentrations in the kidney and the pancreas of rats. The cellular localization of transamidinase was investigated in fetal, neonatal, and adult rat pancreatic tissue using three indicators of the presence of transamidinase: (1) immunofluorescence microscopy, (2) in vitro enzymatic activity measurements on homogenates of whole pancreas and on isolated acinar and islet tissue from adult rats, and (3) ornithine production from perfused adult rat pancreas. The cellular localization of transamidinase was determined in fetal, neonatal, and adult rat pancreas, using a polyclonal guinea pig antibody made against a highly purified preparation of kidney transamidinase. Immunoreactive transamidinase was detected only in the pancreatic acinar cells. The cellular distribution of the immunostaining was compatible with the presence of transamidinase in mitochondria. The transamidinase enzymatic activity of whole pancreatic homogenates was 13.4 +/- 0.7 U/g wet weight (n = 11). In pancreata where islets had been isolated away from the acinar tissue, the transamidinase activity was similar to that of the whole pancreatic homogenates (16.8 +/- 2 U/g wet weight). Any transamidinase activity present in isolated islets was below the sensitivity of the assay. Transamidinase activity in the isolated perfused pancreas was determined by measuring the amount of ornithine released into the perfusate. The transamidinase activity of the perfused pancreas was 16.4 +/- 1.8 U/g pancreas and is an estimate of the physiological production capacity of the enzyme (270 +/- 29 nmol ornithine/min/g pancreas). These results indicate that transamidinase is present at high concentrations in the pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth effects of gastrin in the neonatal rat pancreas: [formula omitted], M Ahmed, PJ Kumar, ML Clark. Digestive Disease Research Centre, St Bartholomew's Hospital, London, U.K.

Growth effects of gastrin in the neonatal rat pancreas: [formula omitted], M Ahmed, PJ Kumar, ML Clark. Digestive Disease Research Centre, St Bartholomew's Hospital, London, U.K.

Appearance of neuropeptides and NADPH-diaphorase during development of the enteropancreatic innervation

Pancreactic ganglia are formed by neural crest-derived precursors, are innervated by enteric neurons, and contain neuropeptides. In addition, the enzyme NADPH-diaphorase is located in a subset of enteric and pancreatic neurons. The expression of neural markers (GAP-43 and NC-1), neurotransmitter-related markers (including neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP), galanin (GAL), dopamine β hydroxylase (DBH), substance P (SP), calcitonin gene-related peptide (CGRP), and NADPH-diaphorase was studied in the fetal and neonatal rat gut and pancreas (E12–P28) in situ and in vitro. NC-1, GAP-43 and DBH-immunoreactive cells were found in the primordial stomach on day E12, and in the pancreas on day E13, along with NPY in endocrine cells. Pancreatic NPY-immunoreactive neurons were detected by day E18. CGRP was seen in the foregut at day E12 but not in the pancreas until day E14. Other neuropeptides (SP, GAL, GRP and VIP) all appeared in the foregut earlier than in the pancreas. NADPH-diaphorase activity was first found in situ in foregut neurons on day E13, and in the pancreas on day E14, but seen in explants a day earlier. These observations show that development of neurons occurs earlier in the gut than in the pancreas, and that NADPH-diaphorase activity appears earlier than the immunoreactivities of the neuropeptides.

Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas.

Cytokeratins (CKs) serve as immunocytochemical markers of epithelial cells. We found that duct cells of the neonatal and adult rat pancreas express CKs 7, 19, and 20. Because pancreatic endocrine cells are thought to derive from duct cells, we examined their expression of CKs 7, 19, and 20 during the neonatal period and the proliferative activity of the different cell types in and around islets. Immunocytochemical analysis revealed that the islets of the neonatal pancreas, in contrast to those of adults, strongly expressed CKs 7, 19, and 20, particularly within a peripheral mantle zone that was continuous with the epithelium of adjacent ductules. This pattern was found during the first 2 weeks of life when significant islet growth occurred as determined by morphometry and bromodeoxyuridine (BrdU) labeling. Moreover, BrdU labeling kinetics indicated that the growth of neonatal islets occurred in the peripheral mantle region characterized by intense CK20 and CK19 immunoreactivity and not in the region composed of differentiated endocrine cells. These observations strongly suggest that proliferating ductal cells associated with islets represent a pool of islet beta- and non-beta-precursor cells.

Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas

Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas

Pancreastatin-Likelmmunoreactivity in the Pancreas of Newborn Rats

Pancreastatin (PST), a novel COOH-terminally alpha-amidated peptide is a part of Chromogranin A molecule. Precursor processing implies also the final amidation step dependent on peptidylglycine alpha-amidating monooxygenase (PAM). High activity of PAM as well as Thyroliberin (TRH, another alpha-amidated peptide) concentration and biosynthesis were reported to be very high in neonatal rat pancreas. We followed the concentration of PST-like immunoreactivity in rat pancreas during ontogenesis. High perinatal PST concentration, resembling that of previously reported for PAM activity and TRH concentration was found. These findings suggest that perinatal PAM activation may affect a broader spectrum of pancreatic peptides.

Enhancement by streptozotocin-induced diabetes of pancreatic prolyl endopeptidase activity in neonatal rats.

The effect of streptozotocin (STZ)-induced diabetes on the concentrations of deamidated TRH (TRH-OH), a metabolite of thyrotropin-releasing hormone (TRH) and prolyl endopeptidase (PE) activity were studied in the pancreas of neonatal rats to determine the contribution of beta-cells to PE activity and TRH-OH levels that we have previously found in this tissue. STZ treatment caused a significant reduction of immunoreactive TRH-OH levels on day-3 and -5 compared to untreated control rats. Reverse-phase high performance liquid chromatography of pooled extracts of 3-day-old normal rat pancreas revealed that about 50% of immunoreactive TRH-OH was found in the fractions representing authentic TRH-OH, whereas the remaining 50% eluted earlier. In STZ treated rats, all of the TRH-OH immunoreactive was associated with this early peak, no authentic TRH-OH could be detected. The specific activity of PE, on the other hand, rose 2.5-fold in diabetic 3-day-old pups (4.06 +/- 0.13 compared with 1.59 +/- 0.83 nmol/min/mg protein, P < 0.01, in controls). This increase declined with age (1.6- and 1.3-fold in 5 day- and 7-day-old pups, respectively). STZ treatment did not change pancreatic PE levels in 20-day-old rats control. Normalization of STZ induced hyperglycemia by sodium metavanadate treatment or by replacement of exogenous insulin did not restore pancreatic PE activity. The enhancement of PE activity following STZ treatment was specific for pancreas tissue. Furthermore, beta cytoxin drugs other than STZ that cause permanent diabetes such as alloxan enhanced PE activity to the same extend. Kinetic studies for PE activity show that Vmax is 3-fold higher in 3-day-old STZ-treated than in rat controls. In contrast, values for Km were comparable in rats of both groups (25 to 34 microM). We then tested whether the decrease of Vmax might have been caused by the presence of an PE inhibiting factor in these preparations. Gel-filtration experiments of pancreatic extracts revealed that the total apparent activity of eluted PE in 3-day-old control rats was 2-fold higher than in the original extract. In contrast, the recovery of eluted PE activity was not increased in the case of STZ-treated 3-day-old and untreated 20-day-old rats. These findings demonstrate that TRH-OH is identified in beta-cells and that an inhibiting factor(s) present in beta-cells appear(s) to be responsible for the unexpected enhancement of PE activity observed in STZ-treated neonatal rats.(ABSTRACT TRUNCATED AT 400 WORDS)