Normal Rat Pancreas Research Articles

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 (10−12–10−6M) 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 (10−12–10−6M) 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 10−6M). 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.

Distributional patterns of phospholipase C isozymes in rat pancreas.

Phospholipase C (PLC) isozymes are believed to play a role in regulating pancreatic exocrine and endocrine secretion. In an attempt to investigate the role of PLC, we examined the distribution patterns of PLC isozymes in the normal rat pancreas by Western blot analysis and immunohistochemistry. Western blot analysis was performed on pancreatic acinar tissues and the islet of Langerhans, which were separated from each other. PLC-beta isozymes (beta1, beta2, beta3, and beta4), delta1, and delta2 were detected in both acinar and islet cells, whereas PLC-gamma1 and gamma2 were observed only in acinar tissues. On immunohistochemistry, the immunoreactivities of PLC isozymes except for PLC-gamma1 were observed as follows: PLC-beta1, in both the exocrine and endocrine tissues; PLC-beta2, mainly in the periphery of the islet and acinar cells; PLC-beta3, in the periphery of the islet and in some ductal epithelium; PLC-beta4, through the islet of Langerhans and ductal epithelium; PLC-gamma1, not detected in pancreatic tissue; PLC-gamma2, mainly in acinar cells; PLC-delta1 and delta2, in the islet and in ductal epithelium. These results suggest that the intrapancreatic site-specific existence of PLC isozymes may modulate pancreatic exocrine and endocrine functions through a PLC-mediated signal transduction.

Site-Specific Localization of Protein Kinase C Isoforms in Rat Pancreas

Background: Protein kinase C (PKC), a major signal-transducing enzyme, is recognized to play an important role in the regulation of pancreatic exocrine and endocrine secretion, and yet the distribution of PKC isoforms in rat pancreas has remained unclarified. Aim of the Study: We examined the precise localization of PKC isoforms to elucidate the role of PKC in the normal rat pancreas. Methods: Male Sprague-Dawley rats were used throughout the experiment. For Western blot analysis, the islet of Langerhans and the acinar tissue were separated by the collagenase digestion method. Also, the whole pancreas was taken out and immunohistochemistry performed. Results: According to Western blot analysis, PKC-α, -γ, -δ, -Ε, -ς, and -λ were detected in both acinar and islet cells while PKC-βII were observed exclusively in the islet. PKC-βI was not observed. On immunohistochemistry, the immunoreactivities of PKC isoforms were observed as follows: PKC-α, weakly in some endocrine cells and ductal epithelium; PKC-βII, mainly in the islet center; PKC-γ, in the islet, intrapancreatic ganglia and ductal epithelium; PKC-δ, in the islet periphery, weakly in some acinar cells and ductal epithelium; PKC-Ε, strongly in the islet, acinar cell and ductal epithelium; PKC-ς, in the islet, acinar cell and ductal epithelium; PKC-λ in some endocrine cells and ductal epithelium. Conclusion: These results suggest that the intrapancreatic site-specific existence of PKC isoforms may regulate pancreatic exocrine and endocrine functions via a PKC-mediated signal transduction.

Activation of pancreatic acinar cells on isolation from tissue: cytokine upregulation via p38 MAP kinase.

Cytokines produced by pancreatic acinar cells may mediate cell death and recruitment of inflammatory cells into pancreas in pancreatitis and other disorders. Here, we demonstrate mRNA expression for a number of cytokines in acini isolated from rat pancreas. Using RNA from microscopically selected individual cells, we confirmed the acinar cell as a source for cytokine expression. Competitive RT-PCR, Western blot analysis, and immunocytochemistry showed large amounts of monocyte chemotactic protein-1 and interleukin-6 compared with other cytokines. Cytokine expression was inhibited by either inhibitors of p38 mitogen-activated protein kinase (MAPK), SB-202190 and SB-203580, or (less strongly) by the transcription factor nuclear factor (NF)-kappaB inhibitor MG-132. A combination of SB-203580 and MG-132 inhibited mRNA expression of all cytokines by >90%. The results suggest a major role for p38 MAPK and involvement of NF-kappaB in cytokine expression in pancreatic acinar cells. In contrast to isolated acini, we detected no or very low cytokine expression in normal rat pancreas. Our results indicate that activation of p38 MAPK, transcription factors, and cytokines occurs during removal of the pancreas from the animal and isolation of acini.

Distribution of NPY and SP and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues☆

Neuropeptides modulate the function of classic neurotransmitters in the regulation of body function. The role of neuropeptides in the regulation of endocrine secretion from the pancreas of diabetic rat is poorly understood. This study examined the pattern of distribution of neuropeptide-Y (NPY) and substance P (SP) in normal and diabetic rat pancreases. In addition to this, the effect of NPY and SP on glucagon secretion was also examined in the pancreases of normal and diabetic rats. Four weeks after the induction of diabetes, the pancreaseses of normal and diabetic rats were removed and processed for immunohistochemistry and glucagon secretion. The pattern of distribution of glucagon in the pancreas of diabetic rat was conspicuously deranged after the onset of diabetes. The pattern of distribution of NPY and SP was, however, similar in the pancreases of both normal and diabetic rats. Stimulation of normal rat pancreatic tissue with NPY (10 −12 and 10 −9 M) evoked large and significant ( P < 0.001) increases in glucagon secretion compared to basal. In contrast to this, NPY inhibited glucagon secretion from the pancreas of diabetic rat. Treatment of pancreatic tissue fragments of normal rat with 10 −9 M SP resulted in significant ( P < 0.03) increases in glucagon secretion. SP inhibited glucagon secretion from diabetic rat pancreas. In conclusion, NPY and SP stimulated glucagon secretion from the pancreas of normal rat. In contrast, NPY and SP inhibited glucagon secretion from diabetic rat pancreas.

Inducible nitric oxide synthetase is expressed in adult but not fetal pig pancreatic islets.

Cytokine-induced expression of inducible nitric oxide synthetase (iNOS) and production of nitric oxide (NO) by pancreatic islet cells has been suggested as one potential mechanism for beta cell destruction. In this study, we investigated the role of iNOS and NO in islet primary non-function. Islets were assessed for their function, viability and expression of iNOS. Adult rat and pig islets isolated by collagenase digestion and fetal pig pancreas (FPP) grafts isolated by collagenase digestion or high oxygen culture were transplanted into C57BL6 mice and nude mice. iNOS protein was detected by immunohistochemistry. iNOS protein was found in normal rat and pig pancreas and adult rat and pig islets that were isolated by collagenase digestion and transplanted into either C57BL6 mice or nude mice. iNOS was not detected in fetal pig islet grafts, regardless of whether collagenase was used in the isolation process. In adult pig islet grafts, the presence of iNOS protein correlated with high levels of islet cell apoptosis and primary non-function. Despite the persistent presence of iNOS in rat islets, there was no evidence that it had a deleterious effect on rat islet viability, or function. Therefore, in isolated adult pig islets, there was a correlation between iNOS expression and apoptosis, suggesting that iNOS activation may be deleterious to the adult pig islets. However, other factors such as the fragility of the islet capsule may be equally important. By contrast, fetal pig islets did not express iNOS and this may be an important reason for their enhanced viability when compared with adult islet tissue.

Distribution of neurotransmitters and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

The distribution of adrenergic, cholinergic and amino acid neurotransmitters and/or their enzymes were examined in both the normal and diabetic pancreatic tissues in rat using immunohistochemistry to determine whether changes in the pattern of distribution of nerves containing these neurotransmitters will occur as a result of diabetes mellitus. In addition to this, the effect of noradrenaline (NA), adrenaline (ADR), acetylcholine (ACh) and γ-amino butyric acid (GABA) on glucagon secretion from the isolated normal and diabetic pancreatic tissues was also investigated. Pancreatic fragments from the tail end of normal and diabetic rats were removed and incubated with different concentrations (10–8–10–4M) of these neurotransmitters. Glucagon secretion into the supernatant was later determined by radioimmunoassay. NA at 10–6M evoked a three-fold increase in glucagon secretion from normal pancreatic tissue fragments. In diabetic pancreatic tissue, NA at 10–6M was able to increase glucagon secretion 1.5 times the value obtained from diabetic basal. ADR (10–8M) increased glucagon secretion slightly but not significantly in normal pancreatic tissue. ADR inhibited glucagon secretion from diabetic pancreas at all concentrations. ACh (10–8M) induced a five-fold increase in glucagon secretion from normal pancreatic tissue. In a similar way, ACh evoked a two-fold increase in glucagon secretion from diabetic pancreas at 10–4M. In normal pancreatic tissue, GABA produced a slight but not significant increase in glucagon secretion at 10–4M. In contrast to this it inhibited glucagon secretion from diabetic pancreatic tissue fragments at all concentrations. In summary, tyrosine hydroxylase- and choline acetyltransferase-positive nerves are equally well distributed in both normal and diabetic rat pancreas. There was an increase in the number of glucagon positive cells and a decrease in the number of GABA-positive cells in diabetic pancreas. NA and ACh have a potent stimulatory effect on glucagon secretion from normal pancreatic tissue fragments, whereas ADR and GABA produced a small but not significant increase in glucagon secretion from normal pancreas. NA and GABA stimulated glucagon secretion from diabetic pancreas. In contrast, ADR and ACh inhibited glucagon secretion from diabetic pancreas. Neurotransmitters vary in their ability to provoke glucagon secretion from either normal or diabetic pancreas.

Gabapentin-Induced Mitogenic Activity in Rat Pancreatic Acinar Cells

Gabapentin induces pancreatic acinar cell tumors in rats through unknown, yet apparently nongenotoxic mechanisms. The primary objective of this study was to determine whether gabapentin acts as a tumor promoter by stimulating acinar cell proliferation in rat pancreas. To this end, indices of pancreatic growth, including increased pancreatic weight, stimulation of acinar cell proliferation, and/or enhanced expression of immediate-early oncogenes were monitored in rats given gabapentin in the diet at 2 g/kg/day for up to 12 months. Rats fed raw soy flour (RSF), a known inducer of pancreatic acinar cell tumors through cholecystokinin-mediated mitogenic stimulation, were used throughout as positive controls. In addition, recent data suggests that gabapentin binds to the alpha(2)delta subunit of a voltage-gated, L-type calcium channel. Because signaling pathways for proliferative processes in pancreatic acinar cells involve intracellular calcium mobilization, the effects of gabapentin on intracellular calcium mobilization ([Ca(2+)](i)) and (3)H-thymidine incorporation were investigated in pancreatic acinar cells isolated from normal rat pancreas and in the AR42J rat pancreatic tumor cell line. As indicated by BrdU labeling indices, acinar cell proliferation increased 3-fold by Day 3 of RSF treatment and remained slightly greater than controls throughout the experiment. Pancreatic weights of RSF-fed rats were 32 to 56% greater than controls throughout the experiment. In contrast, gabapentin had no effect on pancreatic weight or acinar cell labeling index, and therefore had no apparent effect on pancreatic growth. In isolated pancreatic acinar cells, however, gabapentin induced mobilization of intracellular calcium and caused a slight increase in (3)H-thymidine incorporation. The data suggest that gabapentin may possess low level mitogenic activity, which is not easily detectable in in vivo assays.

Distribution of calcitonin-gene-related peptide, neuropeptide-Y, vasoactive intestinal polypeptide, cholecystokinin-8, substance P and islet peptides in the pancreas of normal and diabetic rats

Neuropeptides and peptides are particularly important in the co-ordination of pancreatic exocrine and endocrine secretions. In diabetes mellitus, pancreatic endocrine secretion is particularly impaired. This study investigates whether there is a change in the pattern of distribution of neuropeptides including calcitonin-gene-related peptide (CGRP), neuropeptide-Y (NPY), vasoactive intestinal polypeptide (VIP), cholecystokinin-octapeptide (CCK-8), substance P (SP), and islet peptides including insulin (INS), glucagon (GLU), somatostatin (SOM) and pancreatic polypeptide (PP) in the pancreas of streptozotocin (STZ)-diabetic rats. After the onset of diabetes, the pattern of distribution of INS, GLU, SOM and PP cells was deranged. CGRP was demonstrated in ganglion cells of both normal and diabetic pancreas. CGRP was also localized in nerve fibres innervating the blood vessels of both normal and diabetic pancreas. The pancreata of both normal and diabetic rats contained numerous NPY-immunopositive varicose nerve fibres in the wall of blood vessels. In normal pancreatic tissue, VIP-immunopositive nerve fibres were observed in all areas of the pancreas. After the onset of diabetes, VIP-positive nerve fibres were still discernible in the interacinar regions of the pancreas. CCK-8 was identified in nerve fibres innervating both the normal and diabetic rat pancreata. These CCK-8-immunopositive nerves were varicose in nature and distributed in the wall of blood vessels. SP was demonstrated in neurons located in the interlobular areas of normal tissue and in fine varicose nerve fibres of the interacinar region of STZ-induced diabetic pancreas. In conclusion, CGRP, NPY, VIP, CCK-8 and SP are well distributed in both normal and diabetic pancreas.

Glucose-dependent insulinotropic effects of JTT-608, a novel antidiabetic compound

The effects of JTT-608 [ trans-4-(4-methylcyclohexyl)-4-oxobutyric acid], a novel antidiabetic compound, on insulin secretion were investigated using mouse insulinoma cell line (MIN6 cells) and isolated, perfused rat pancreas. JTT-608 enhanced insulin secretion in MIN6 cells in a dose dependent (10–300 μM) and glucose concentration-dependent (2.8–16.7 mM) manner. Unlike sulphonylureas, JTT-608 minimally stimulated insulin secretion at low glucose concentrations but potently enhanced insulin secretion at high glucose concentrations. In isolated, perfused pancreas of normal rats, JTT-608 (100–300 μM) dose-dependently enhanced insulin secretion in the first and second phases at high glucose concentrations but minimally stimulated insulin secretion at a basal glucose concentration. In isolated, perfused pancreas of neonatally streptozotocin-induced non-insulin-dependent diabetes mellitus rats (nSTZ rats), JTT-608 (200 μM) normalized the first phase and doubled the second phase of insulin secretion. In MIN6 cells, JTT-608 did not inhibit the binding of [ 3 H ]glibenclamide to membrane fractions but enhanced K +-ATP channel-independent insulin secretion. These results suggest that JTT-608 enhances insulin secretion in a different manner and via a different mechanism from hypoglycemic sulphonylureas.

Immunohistochemical Characterization of Pancreatic Tumors Induced by Dimethylbenzanthracene in Rats

Dimethylbenzanthracene (DMBA) induces pancreatic adenocarcinomas in rats 9 months after carcinogen exposure, with precursor lesions (tubular complexes) developing 1 month after initiation of treatment. Because previous studies have suggested an acinar cell of origin for these tumors, we investigated the expression pattern of ductal, acinar, and islet cell markers in these cancers to gain insight into their phenotype and cell of origin. Pancreatic neoplasms were induced in rats by implantation of DMBA into the head of the pancreas. Lesions studied included 10 early tubular complexes (DMBA for 2 weeks), 8 tubular complexes (DMBA for 1 month), and 10 adenocarcinomas (DMBA for 9 months). Normal rat pancreas served as a control. For comparison, 5 human ductal adenocarcinomas were also evaluated. Immunohistochemistry with ductal (keratin, cytokeratin 19, cytokeratin 20), acinar (chymotrypsin), and islet (chromogranin A) cell markers was performed to analyze the tissues. Rat tubular complexes and adenocarcinomas revealed strong expression of keratin, cytokeratin 19, and cytokeratin 20 in the cytoplasm of all neoplastic cells, absence of chymotrypsin, and rare immunoreactivity to chromogranin A. Human adenocarcinomas showed strong expression of keratin and cytokeratin 19 in all neoplastic cells, expression of cytokeratin 20 in 5-20% of cells, and absence of chymotrypsin and chromogranin A. Pancreatic adenocarcinomas induced by DMBA in rats express markers consistent with a ductal phenotype, as observed in human tumors. Ductal marker expression in early tumor stages suggests a ductal cell of origin.

Interaction of islet hormones with cholecystokinin octapeptide-evoked secretory responses in the isolated pancreas of normal and diabetic rats.

This study investigates the effects of the islet hormones, insulin (Ins), glucagon (Glu) and somatostatin (Som) with cholecystokinin octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca2+]i and their pattern of distribution in the isolated pancreas of normal and diabetic rats. Ins and Glu evoked small increases in amylase output from pancreatic segments compared with a much enhanced effect of CCK-8. In contrast, Som induced a biphasic response comprising an initial decrease followed by a secondary increase and this biphasic response may be dependent upon the concentration. Combining the islet hormones with CCK-8 resulted in marked potentiation in amylase output compared with either CCK-8 alone or the individual hormone. Genistein and tyrphostin A25, the tyrosine kinase inhibitors, evoked a small decrease in amylase output from pancreatic segments. They had no effect on the CCK-8-evoked secretory response but markedly inhibited the potentiation of the islet hormones with CCK-8. In pancreatic acini and acinar cells Ins, Glu and Som individually evoked small increases in amylase output compared with a much larger response with CCK-8. When the islet hormones were combined with CCK-8 there was no potentiation of amylase output. Similarly, when rats were rendered diabetic by prior treatment with streptozotocin Ins, Glu and Som failed to potentiate the secretory response of CCK-8. In fura-2-loaded pancreatic acinar cells Ins or Glu evoked small increases in [Ca2+]i compared with a much larger elevation with CCK-8. Ins, Glu and Som each enhanced the CCK-8-evoked [Ca2+]i. Genistein elicited a decrease in [Ca2+]i both in the absence and presence of the islet hormones. It also decreased the elevation in [Ca2+]i resulting from the combined presence of CCK-8 with either Ins or Glu but it had no effect on CCK-8 in combination with Som. In pancreatic acinar cells from diabetic rat Ins, Glu and Som had no detectable effect on CCK-8-evoked elevation in [Ca2+]i compared with the response obtained with CCK-8 alone. CCK-8-immunopositive cells were distributed around the walls of blood vessels, numerous Ins-positive cells in the central and peripheral parts of the islets of Langerhans, Glu-immunoreactive cells in the periphery of islets and Som-positive cells in the outer part of the islets. During diabetes, the number of CCK-immunopositive cells remained unchanged whereas the number of Ins-positive cells decreased coupled with an increase in the number of Glu-positive cells. The results indicate that both tyrosine kinase and cellular Ca2+ seem to be the intracellular mediators involved with the enhanced secretory responses obtained with a combination of the islet hormones with CCK-8. Moreover, the presence of viable pancreatic islets of Langerhans seems to be associated with the potentiation of the islet hormones with CCK-8.

Interaction of islet hormones with cholecystokinin octapeptide-evoked secretory responses in the isolated pancreas of normal and diabetic rats

This study investigates the effects of the islet hormones, insulin (Ins), glucagon (Glu) and somatostatin (Som) with cholecystokinin octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca2+]i and their pattern of distribution in the isolated pancreas of normal and diabetic rats. Ins and Glu evoked small increases in amylase output from pancreatic segments compared with a much enhanced effect of CCK-8. In contrast, Som induced a biphasic response comprising an initial decrease followed by a secondary increase and this biphasic response may be dependent upon the concentration. Combining the islet hormones with CCK-8 resulted in marked potentiation in amylase output compared with either CCK-8 alone or the individual hormone. Genistein and tyrphostin A25, the tyrosine kinase inhibitors, evoked a small decrease in amylase output from pancreatic segments. They had no effect on the CCK-8-evoked secretory response but markedly inhibited the potentiation of the islet hormones with CCK-8. In pancreatic acini and acinar cells Ins, Glu and Som individually evoked small increases in amylase output compared with a much larger response with CCK-8. When the islet hormones were combined with CCK-8 there was no potentiation of amylase output. Similarly, when rats were rendered diabetic by prior treatment with streptozotocin Ins, Glu and Som failed to potentiate the secretory response of CCK-8. In fura-2-loaded pancreatic acinar cells Ins or Glu evoked small increases in [Ca2+]i compared with a much larger elevation with CCK-8. Ins, Glu and Som each enhanced the CCK-8-evoked [Ca2+]i. Genistein elicited a decrease in [Ca2+]i both in the absence and presence of the islet hormones. It also decreased the elevation in [Ca2+]i resulting from the combined presence of CCK-8 with either Ins or Glu but it had no effect on CCK-8 in combination with Som. In pancreatic acinar cells from diabetic rat Ins, Glu and Som had no detectable effect on CCK-8-evoked elevation in [Ca2+]i compared with the response obtained with CCK-8 alone. CCK-8-immunopositive cells were distributed around the walls of blood vessels, numerous Ins-positive cells in the central and peripheral parts of the islets of Langerhans, Glu-immunoreactive cells in the periphery of islets and Som-positive cells in the outer part of the islets. During diabetes, the number of CCK-immunopositive cells remained unchanged whereas the number of Ins-positive cells decreased coupled with an increase in the number of Glu-positive cells. The results indicate that both tyrosine kinase and cellular Ca2+ seem to be the intracellular mediators involved with the enhanced secretory responses obtained with a combination of the islet hormones with CCK-8. Moreover, the presence of viable pancreatic islets of Langerhans seems to be associated with the potentiation of the islet hormones with CCK-8.

Effect of Zinc on Experimental Acute Pancreatitis in Rats.

The effects of zinc on acute pancreatitis were investigated in rats with cerulein-induced pancreatitis and taurocholate+trypsin-induced pancreatitis. The endogenous zinc concentrations in the serum and pancreas after the onset of acute pancreatitis was not different from those in normal rats. Orally administered zinc sulfate was well absorbed and was taken up by the pancreas in normal rats as well as in rats suffering from acute pancreatitis. Oral administration of zinc sulfate before induction of acute pancreatitis reduced the serum amylase activity and the wet weight of the pancreas in rats treated with cerulein or taurocholate+trypsin. Oral administration of zinc sulfate after the induction of acute pancreatitis also reduced the pancreatic wet weight in rats with cerulein-induced pancreatitis, and decreased the mortality rate in rats with taurocholate+trypsin-induced pancreatitis. N-(3-aminopropionyl)-L-histidinato zinc (polaprezinc), a chelate of zinc and L-carnosine, also decreased the serum amylase activity and pancreatic wet weight in both types of pancreatitis. These data suggest that zinc compounds may have a therapeutic effect on acute pancreatitis.

Loss of alpha 1 connexin does not alter the prenatal differentiation of pancreatic beta cells and leads to the identification of another islet cell connexin.

Connexin alpha 1, also referred to as Cx43, has thus far been the only gap junction protein identified between the hormone-producing cells of pancreatic islets. To investigate whether loss of this connexin affects the development of endocrine pancreas and the differentiation of insulin-producing beta cells, we have taken advantage of a transgenic line in which the gene coding for connexin alpha 1 had been functionally deleted by homologous recombination. Analysis of pancreas at embryonal day 19.5 (E 19.5) after immunostaining for the four main types of islet hormones, showed that islet cell development was similar in homozygous transgenic mice that completely lacked alpha 1 connexin, in mice that were heterozygous for the transgene, and in age-matched controls with a genetic background similar to that of the transgenic animals. In particular, the three animal groups featured beta cells that had a similar insulin content and ultrastructural organization, including the presence of typical gap junction plaques on the membrane. However, quantitative analysis of freeze-fractured membranes showed that these plaques were less frequent in the transgenic mice lacking alpha 1 connexin. This finding prompted us to revisit the connexin pattern of normal pancreatic beta cells. Using RT-PCR amplification and primers specific for nine of the mammalian connexins, we have found that normal rat and mouse pancreas contain six connexin transcripts, including one that codes for alpha 6 connexin, a protein also referred to as Cx45. This transcript was also identified in isolated pancreatic islets, in FACS-purified suspensions of primary beta cells and in the insulin-producing cells of an experimental tumor. Using antibodies, we found that connexin alpha 6 is expressed by the latter cells, as well as by pancreatic fibroblasts and epithelial duct cells. The data show that pancreatic islets have a normal prenatal development in mice that no longer express alpha 1 connexin. They further provide evidence that normal and tumoral insulin-producing cells natively coexpress connexins alpha 1 and alpha 6.

Loss of α1 connexin does not alter the prenatal differentiation of pancreatic β cells and leads to the identification of another islet cell connexin

Connexin α1, also referred to as Cx43, has thus far been the only gap junction protein identified between the hormone-producing cells of pancreatic islets. To investigate whether loss of this connexin affects the development of endocrine pancreas and the differentiation of insulin-producing β cells, we have taken advantage of a transgenic line in which the gene coding for connexin α1 had been functionally deleted by homologous recombination. Analysis of pancreas at embryonal day 19.5 (E19.5) after immunostaining for the four main types of islet hormones, showed that islet cell development was similar in homozygous transgenic mice that completely lacked α1 connexin, in mice that were heterozygous for the transgene, and in age-matched controls with a genetic background similar to that of the transgenic animals. In particular, the three animal groups featured β cells that had a similar insulin content and ultrastructural organization, including the presence of typical gap junction plaques on the membrane. However, quantitative analysis of freeze-fractured membranes showed that these plaques were less frequent in the transgenic mice lacking α1 connexin. This finding prompted us to revisit the connexin pattern of normal pancreatic β cells. Using RT-PCR amplification and primers specific for nine of the mammalian connexins, we have found that normal rat and mouse pancreas contain six connexin transcripts, including one that codes for α6 connexin, a protein also referred to as Cx45. This transcript was also identified in isolated pancreatic islets, in FACS-purified suspensions of primary β cells and in the insulin-producing cells of an experimental tumor. Using antibodies, we found that connexin α6 is expressed by the latter cells, as well as by pancreatic fibroblasts and epithelial duct cells. The data show that pancreatic islets have a normal prenatal development in mice that no longer express α1 connexin. They further provide evidence that normal and tumoral insulin-producing cells natively coexpress connexins α1 and α6. Dev. Genet. 24:13–26, 1999. © 1999 Wiley-Liss, Inc.

Effects of islet hormones on nerve-mediated and acetylcholine-evoked secretory responses in the isolated pancreas of normal and diabetic rats.

This study employs the pancreas of normal and diabetic rats to investigate the relationship between the endocrine and exocrine pancreas in the control of exocrine secretion employing enzyme and immunohistochemical and physiological techniques. Acetylcholine esterase (ACh-E) positive nerves were distributed in the interacinar regions of the pancreas lying close to the exocrine cells. There was no difference between the cholinergic innervation of the pancreas in normal and diabetic rat. Insulin (INS) immunopositive cells were observed in the peripheral and central portions of the Islet of Langerhans in the pancreas of normal rat. In the diabetic animals the number of INS-positive cells were decreased. In contrast, glucagon (GLU) and somatostatin (SOM)-immunopositive cells were identified mainly in the peripheral parts of the Islets of Langerhans and their numbers increased markedly in the diabetic pancreas. Insulin alone had no significant effect on amylase secretion in the normal pancreas whereas GLU and SOM evoked small increases in amylase out compared to basal. In contrast, the islet hormones have no detectable secretory effect on the diabetic pancreas compared to control. Both electrical field stimulation (EFS) of intrinsic secretomotor nerves and exogenous application of acetylcholine (ACh) resulted in marked increases in amylase secretion. In pancreatic acini and acinar cells ACh evoked dose-dependent increases in amylase release. In normal pancreatic segments a combination of either INS or GLU with EFS or ACh resulted in marked potentiation of amylase output. In contrast, SOM inhibited the EFS-evoked amylase output but enhanced the secretory response to ACh. In pancreatic acini and acinar cells from normal rat and in pancreatic segments from diabetic rats, the islet hormones had no potentiating effect on the ACh-evoked secretory response. Similarly, in the diabetic rat the islet hormone had no effect on EFS-evoked amylase output. In fura-2 loaded pancreatic acinar cells ACh-induced a marked increase in intracellular free calcium concentration [Ca2+]i compared to basal. Either INS or GLU, but not SOM, elicited a small increase in [Ca2+]i. Combining either INS or GLU with ACh resulted in a potentiation of [Ca2+]i compared with ACh alone. In contrast, SOM had no significant effect on the ACh-induced [Ca2+]i compared to the response obtained with ACh alone. In pancreatic acinar cells of diabetic rat ACh-elicited similar magnitude of [Ca2+]i compared to acinar cells of normal rat. However, when the islet hormones were combined with ACh there was no enhancement of [Ca2+]i compared to ACh alone. The results indicate that the potentiation of either EFS or ACh-evoked secretory responses by the islet hormones seem to occur only in pancreatic segments which have intact viable Islets of Langerhans and not in either acini and acinar cells or from the pancreas of diabetic rat. Moreover, it is apparent that cellular Ca2+ is involved with the interaction of ACh with either INS or GLU.

Effect of chronic oral administration of the CCK receptor antagonist loxiglumide on exocrine and endocrine pancreas in normal rats.

In normal adult rats, administration of a low dose of loxiglumide for 7 d had no significant effect on exocrine and endocrine pancreatic function, whereas a high dose of loxiglumide decreased pancreatic enzyme output without inducing insulin resistance and diabetes mellitus. There is a possibility that chronic administration of cholecystokinin receptor antagonists not only inhibits the growth of the pancreas but also alters exocrine and endocrine pancreatic function. Loxiglumide at a dose of 50, 100, or 200 mg/kg body weight, or the same volume of saline, was given by an orogastric tube twice daily for 7 d (13 successive times). Biochemical and functional changes were determined on d 8 at 24 h after the last administration of loxiglumide and 18 h fasting. Pancreatic exocrine and endocrine function was simultaneously determined following an intravenous injection of a mixed solution of 0.5 g/kg body weight glucose plus 100 ng/kg body weight cerulein. Pancreatic weight and protein content were dose-dependently decreased by loxiglumide, whereas DNA content was decreased only by the highest dose of loxiglumide. Loxiglumide caused dose-dependent decreases in pancreatic fluid and protein output. Total pancreatic insulin content in rats treated with loxiglumide was not significantly different from that in the control rats. However, insulin concentration relative to DNA content was significantly increased in rats treated with 200 mg/kg body weight loxiglumide compared with that in other groups of rats. Glucose-stimulated insulin release was significantly low in rats treated with the highest dose of loxiglumide compared with that in other groups of rats, although there was no difference of serum glucose concentrations among these four groups of rats.

Modulation of gliquidone action by forskolin in the pancreas of normal and GK rats.

This study aims to investigate whether agents that stimulate adenosine 3',5'-cyclic monophosphate (cAMP) formation could be used to increase insulin release evoked by hypoglycemic sulfonylureas in non-insulin-dependent diabetes mellitus. For this purpose, the effect of gliquidone (1.0 microM) on insulin and glucagon release was examined in the perfused pancreas of either normal or Goto-Kakizaki (GK) rats at a low concentration of D-glucose (2.8 mM) and in the absence or presence of forskolin (1.3 microM). In normal rats, the diterpene exerted relatively little effect on basal insulin release but markedly augmented the insulinotropic action of gliquidone. In GK rats, forskolin dramatically augmented both basal and gliquidone-stimulated insulin output. In mirror image of its effect on insulin release, forskolin augmented basal glucagon output and failed to increase the secretory response of the A cell to gliquidone, at least in normal rats. On the contrary, in GK rats, forskolin, while slightly enhancing basal glucagon output, unmasked the glucagonotropic potential of gliquidone that was otherwise not detected in the diabetic animals. These findings are interpreted in light of a dual metabolic and energy-independent response of islet cells to the forskolin-induced generation of cAMP. They document the optimalization by endogenous cAMP of the B cell secretory response to gliquidone in non-insulin-dependent diabetes.

Models of exocrine pancreatic pathologies: a microscopical point of view.

The objective of this review is to emphasize the contribution of optical and electronic microscopy to the study of the normal and pathological rat pancreas. These basic techniques used to explore the pancreatic morphology are capital to precisely localize specific enzymes or proteins in cellular subcompartments, to establish the normal or the pathological state of the gland, and to detect and describe unusual structures that appear under certain experimental conditions.