Cellular and subcellular localization of transforming growth factor-alpha and epidermal growth factor receptor in normal and diseased human and hamster pancreas

Four normal pancreas, 8 chronic pancreatitis specimens, and 30 non-endocrine pancreatic tumors from humans and 6 normal and 6 induced pancreatic cancers in hamsters were examined immunohistochemically by antibodies against human transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR). Two normal pancreas and two pancreatic cancer specimens from each species were also studied immunoelectron microscopically by the immunogold method. In chronic pancreatitis, the reactivity and intensity of the staining with both antibodies were much greater in ductal/ductular cells than in the normal pancreas. All 30 pancreatic cancers reacted with both antibodies with a variable degree of reactivity and staining intensity. No correlation was found between the histological type of tumors, the degree of tumor differentiation, and the incidence and patterns of reactivity of either antibody. Immunoelectron microscopically, both EGFR and TGF-alpha were demonstrated primarily on the basal membrane. In the normal hamster pancreas, TGF-alpha was overexpressed in the alpha-cells but not in any other islet cells. Both TGF-alpha and EGFR were marginally detectable in the exocrine pancreas and in induced pancreatic lesions. This is the first demonstration of subcellular localization of TGF-alpha and EGFR in the normal and diseased human and hamster pancreas.     

Experimental evidence for the origin of ductal-type adenocarcinoma from the islets of Langerhans

To investigate the role of the islets of Langerhans in pancreatic carcinogenesis, freshly isolated islets from male Syrian hamsters were transplanted into the right submandibular glands of 50 female hamsters that were or were not pre-treated with streptozotocin. Thyroid gland fragments, cellulose powder, and immortal hamster pancreatic ductal cells were injected into the left submandibular gland of the same hamsters. All recipient hamsters were then treated with the potent pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine weekly at a dose of 40 mg/kg of body weight for 3 weeks. Between 3 and 8 weeks later, 18 of 75 (24%) hamsters developed large ductal-type adenocarcinomas in the submandibular gland region, where islets were transplanted, but none developed tumors in the left submandibular gland. In 9 of 18 hamsters, tumors were multiple so that a total of 31 cancers were found. Eleven of these carcinomas were in the vicinity of transplanted islets, eight of which showed intra-insular ductular or cyst formation as seen in the pancreas of hamsters during pancreatic carcinogenesis. The formation of ductular structures within islets was also demonstrated in vitro. Some tumor cells in the vicinity of these islets were reactive with anti-insulin. Y chromosome message was found by polymerase chain reaction analysis in one of the three tumors examined. Also, like the induced pancreatic tumors, all three submandibular gland tumors that were examined had the mutation of the c-Ki-ras oncogene at codon 12 and all tumors expressed blood group A antigen. These and other findings strongly suggest that some components of islets, most probably stem cells, are the origin of ductal-type adenocarcinomas in this model.     

Malignant transformation of duct-like cells originating from acini in transforming growth factor transgenic mice

BACKGROUND & AIMS: In transgenic mice overexpressing transforming growth factor (TGF)-alpha in the exocrine pancreas, progressive pancreatic fibrosis and a transdifferentiation of acinar cells to duct-like cells occurs. The present study was undertaken to analyze this transdifferentiation process. METHODS: Pancreatic specimens were characterized using light microscopy and immunohistochemistry. Expression of the epidermal growth factor receptor (EGFR) and TGF-alpha was evaluated with slot blot and Western analysis. To identify other generic events, K-ras mutations were screened with an enriched polymerase chain reaction approach and p53 expression was detected with immunohistochemistry. RESULTS: Morphological examination revealed an aggregation of interlobular fibroblasts and a decrease in acinar cell height starting at day 14 after birth. In older animals, these acinar cells change to duct-like cells, which form tubular structures and express ductal markers. Evidence for dysplastic changes was found in 12 of 21 TGF-alpha transgenic mice older than 1 year. We also observed four malignant pancreatic tumors, which were multicentric and originated from dysplastic tubular complexes. They displayed a mixed cystic-papillary phenotype strongly positive for carbonic anhydrase activity. EGFR expression progressively increased in the transition from acinar to duct-like and transformed cells. Activating K-ras mutations could not be detected; however, tubular complexes and tumors displayed increased immunoreactivity for nuclear p53. CONCLUSIONS: These data suggest an involvement of the TGF-alpha/EGFR pathway in conjunction with other yet unknown events in pancreatic tumor development. Furthermore, these observations are in favor of an acinar-ductal carcinoma sequence. Thus, these transgenic animals will be useful to define genetic alterations associated with a transition from acinar cells to a neoplastic ductal phenotype.     

Inhibitory effect of sarcophytol A on pancreatic carcinogenesis after initiation by N-nitrosobis(2-oxypropyl)amine in Syrian hamsters

Sarcophytol A (SaA), a cembrane-type diterpene, inhibits pancreatic carcinogenesis induced by N-nitrosobis(2-oxypropyl)amine (BOP) in hamsters. The experimental groups received two injections of BOP at 70 mg/kg dose, followed 2 weeks later by a 20 mg/kg dose injection, and were fed a basal diet or 0.01 and 0.05% SaA diets starting 1 week after the second injection of BOP. Control groups were injected with normal saline and fed the basal diet or the 0.05% SaA diet. All animals were killed 30 weeks after the start of the experiments. Seventeen BOP-treated hamsters fed the basal diet developed pancreatic tumors (77.3%) while only 12 of 21 hamsters fed the 0.01% SaA diet (57.1%) and 12 of 23 hamsters fed the 0.05% SaA diet (52.2%) developed pancreatic tumors. Pancreatic lesions included ductal hyperplasia, atypical ductal hyperplasia, and carcinoma in situ. Microscopic invasive carcinoma induced by BOP and the incidence of larger pancreatic tumors in hamsters were significantly higher in hamsters fed the basal diet than in hamsters fed the SaA diet (p < 0.05). The proliferating cell nuclear antigen (PCNA) labeling index of pancreatic carcinoma in BOP-treated hamsters fed the basal diet was 41.2 +/- 13.4%, whereas BOP-treated hamsters fed 0.01 and 0.05% SaA diets yielded PCNA indexes of 26.8 +/- 8.3 and 28.4 +/- 7.0%, respectively. k-ras mutation was detected in 40% of cancers in both groups. No pancreatic tumors developed in saline-treated groups, and no differences in body weights or histological findings in their organs, including the pancreas, were observed in either group. These findings suggest that SaA not only inhibits BOP-induced pancreatic carcinogenesis in hamsters, but also provides antipromotion and antiprogression effects on these tumors, even when SaA commences 1 week after the initiation of pancreatic carcinogenesis.     

Effects of cigarette smoke on N-nitrosobis(2-oxopropyl)amine-induced pancreatic and respiratory tumorigenesis in hamsters

Influences of cigarette smoke on N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic duct and respiratory tract tumorigenesis were investigated using a hamster two-stage carcinogenesis model. Male 5-week-old hamsters were divided into 5 groups. Group 1 was s.c. injected with BOP at a dose of 10 mg/kg once a week for 3 weeks as an initiation treatment together with cigarette smoke exposure over the same 4-week period. Group 2 was exposed to cigarette smoke for 26 weeks after the BOP-initiation. Groups 3 and 4 were respectively given the BOP-initiation alone and the 26-week cigarette smoke exposure without initiation. Group 5 served as a sham-smoked negative control. The experiment was terminated 30 weeks after the first BOP injection. The incidence of pancreatic adenocarcinomas was significantly decreased in Group 1 as compared to the Group 3 value (P < 0.01) while the Group 2 value did not show any change. In contrast, the incidence of laryngeal and tracheal proliferative lesions (hyperplasias and papillomas) was significantly increased in Group 2 over Group 3 (P < 0.01). The incidence of pulmonary hyperplasias was also increased in Group 2 over Group 3 (P < 0.05), although that of pulmonary adenomas or adenocarcinomas was decreased in Group 2 as compared to the Group 3 value (P < 0.01). Cigarette smoke exposure in the BOP-initiation phase (Group 1) did not affect the development of respiratory proliferative lesions. No animals in Groups 4 and 5 developed any tumors in the pancreas or respiratory tract. Our results thus indicate that cigarette smoke exposure inhibits pancreatic carcinogenesis when given in the initiation phase, whereas it modulates (enhances or suppresses) the development of proliferative lesions in the respiratory tract if applied during the promotion stage to hamsters pretreated with BOP.     

VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta-cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP-2 and cellubrevin, but not VAMP-1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta-cells contain secretory granules that store and secrete insulin as well as synaptic-like microvesicles carrying gamma-aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP-2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP-2 with insulin-containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta-cells. Pretreatment of streptolysin-O permeabilized insulin-secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP-2 and cellubrevin and abolished Ca(2+)-induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S-stimulated insulin secretion. Taken together, our results show that pancreatic beta-cells express VAMP-2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)-mediated insulin secretion.     

Factors affecting use of contraception in Matlab, Bangladesh

We examined the effect of voluntary physical exercise (running wheels) on pancreatic carcinogenicity of N-nitrosobis-(2-oxopropyl) amine (BOP) in groups of female Syrian hamsters fed a high-fat (HF) diet in which corn oil was 24.6% of the diet or a low-fat (LF) diet in which corn oil was 4.5% of the diet. Each group was divided into an exercising (EX) group (LF-EX and HF-EX) and a sedentary (S) group (LF-S and HF-S). All hamsters were treated with BOP (20 mg/kg body wt) weekly for two weeks beginning four weeks after the experimental diets, which were fed from weaning. A modified glucose tolerance test was performed before the BOP injections and then again at 20 and 40 weeks, and the levels of glucose, insulin-like growth factor I, and insulin were determined in the plasma samples. At the end of the experiment, serum levels of lipid metabolites were also examined in six hamsters from each group. The experiment was terminated 44 weeks after the BOP treatment. Pancreatic ductal/ductular adenocarcinoma incidence was significantly higher in hamsters fed the HF diet (HF-S and HF-EX) than in those fed the LF diet (LF-S and LF-EX). In all groups, glucose and insulin-like growth factor I levels remained within the normal range throughout the experiment, whereas insulin and lipid metabolite levels were significantly elevated in all hamsters fed the HF diet (HF-S and HF-EX). Exercise significantly reduced the insulin level in the group fed the HF diet but did not influence the cancer burden, possibly by the generation of reactive lipid metabolites. Overall, the results showed that voluntary physical exercise does not influence the promotional action of the HF diet on pancreatic carcinogenesis in hamsters. This action could be attributed to the ability of the HF diet to increase the secretion of insulin, which has a growth-promoting and mitogenic effect on pancreatic cells, and to the effect of an HF diet or physical exercise in producing excess free radicals.     

Dietary energy restriction does not inhibit pancreatic carcinogenesis by N-nitrosobis-2-(oxopropyl)amine in the Syrian hamster

Dietary energy restriction was previously shown to be effective in preventing a wide range of experimentally induced cancers. Studies were conducted to assess the influence on pancreatic carcinogenesis of dietary energy restrictions (reduced fat and carbohydrate) of 10%, 20% or 40% in comparison with control in Syrian hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Two carcinogenesis studies were conducted. One used a single treatment with 20 mg BOP/kg body weight and followed hamsters for 102 weeks following treatment, and the other used three weekly treatments of 20 mg BOP/kg body weight and followed hamsters for 45 weeks after treatment. Hamsters were fed control or energy restricted diet beginning the week following the last BOP treatment. Pancreatic carcinomas were induced in 9-18% of the hamsters in the first experiment and in 59-66% of the animals in the second. Dietary energy restriction did not influence carcinoma incidence in either study, and in the second experiment the multiplicity of tumors was higher in the 40% energy restriction (ER) group than in control hamsters. Plasma corticosterone was suppressed by BOP treatment, particularly in the 20% and 40% ER hamsters in the second experiment, and diet or BOP treatment did not significantly alter plasma cortisol. Pancreatic protein kinase Czeta measured by Western blot was highest in the cytosol and particulate fractions of the 40% ER hamsters in the first experiment. These results indicate that dietary energy restriction is not effective in the prevention of BOP induced pancreatic carcinogenesis in the Syrian hamster.     

Chloroquine, quinine and quinidine inhibit calcium release from macrophage intracellular stores by blocking inositol 1,4,5-trisphosphate binding to its receptor

Endocrine and exocrine pancreatic morphogenesis is known to occur from ductal epithelium, but the factors that regulate this process are unknown. Vascular endothelial growth factor (VEGF)/vascular permeability factor has recently been reported to affect fetal islet ontogenesis. VEGF is an angiogenic factor with a growth-promoting effect that is thought to be restricted to vascular endothelial cells. We demonstrated that VEGF is also a mitogen for adult rat pancreatic duct epithelial cells in primary culture. VEGF supplementation to a serum-free culture medium increased the 5-bromo-2'-deoxyuridine-pulse labeling index of ductal cells more than 2-fold. Immunohistochemical staining and protein blots revealed that pancreatic duct cells express fetal liver kinase-1 high-affinity receptors for VEGF. In pancreatic tissue, immunohistochemistry shows that VEGF peptide is expressed in normal pancreatic islet cells. In duct ligation-induced acute pancreatitis, numerous inflammatory leukocytes containing VEGF were seen to infiltrate between hyperplastic ducts. In the latter model, islet neogenesis has previously been observed. Our data indicate the possibility that VEGF plays a role in the paracrine regulation of ductal growth and differentiation in vivo, eg, in pancreatitis. In vitro, however, VEGF did not induce endocrine differentiation of ductal cells, indicating that it is not the only factor required for the activation of islet neogenesis.     

Histologic and biologic patterns of microscopic pancreatic ductal adenocarcinomas detected incidentally at autopsy

BACKGROUND: The major clinical problems with pancreatic carcinoma are its silent course and late, fatal clinical manifestation. The results of treatments of small pancreatic carcinomas (<2 cm in greatest dimension) have led to the assumption that the detection of these cancers at earlier stages would lead to better survival and possible cure. Currently, there is no information about the histologic and biologic patterns of early stage pancreatic carcinoma, and the available data on incidentally detected tumors are fragmentary. The authors observed two incidental microscopic pancreatic ductal adenocarcinomas in female patients who died of advanced gastric carcinoma (Case 1) and renal carcinoma (Case 2). METHODS: The pancreatic lesions were examined histologically in serial sections and immunocytochemically for islet cells. Microdissection was performed so that the lesions could be examined for c-Ki-ras mutation. RESULTS: In Case 1, the pancreatic lesion was composed of cystic and solid components. The cystic component consisted of four small cysts compatible with a mucinous cystic tumor and showed no invasion. The solid component was a well-differentiated adenocarcinoma that occupied a 4 x 2 mm area. In Case 2, the pancreatic lesion contained two small, separate cysts, one of which was surrounded by two apparently separate, invasive adenocarcinomas 2.6 x 0.7 mm and 1.2 x 0.5 mm in greatest dimension. There was invasion of pancreatic islets and perineural spaces in both cases; and in Case 2, there was invasion of peripancreatic fatty tissue. In both cases, the epithelia of the cystic components and tumors showed mutation of the c-Ki-ras oncogene at codon 12, with GGT-to-GAT transition. CONCLUSIONS. Pancreatic carcinoma seems to occur under occult circumstances and maintain a silent course. Even in its early developmental stage, the cancer is invasive, primarily affects islets and nerves, and exhibits mutation of the c-Ki-ras oncogene. These findings call for urgency in the development of preventive modalities.     

Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles in insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet beta-cells express low molecular weight secretory phospholipases A2 (PLA2) and a Group VI, Ca2+-independent PLA2 (iPLA2). Previous studies indicate that islets also express a protein recognized by antibodies against a Group IV, cytosolic, Ca2+-dependent PLA2 (cPLA2). To further examine the possible expression of cPLA2 by islets, we screened a rat islet cDNA library with a probe that recognizes cPLA2 sequence, and isolated a full-length cPLA2 cDNA. The rat islet cPLA2-deduced amino acid sequence is 96% identical to those of human and mouse cPLA2. Transfection of COS-7 cells with cPLA2 cDNA in an expression vector induced expression of Ca2+-dependent PLA2 activity and of a protein recognized by anti-cPLA2 antibody. Comparison of recombinant islet cPLA2 and iPLA2 activities expressed in transfected COS-7 cells indicated that iPLA2 but not cPLA2 is stimulated by ATP. Both activities are similarly sensitive to inhibition by arachidonyltrifluoromethyl ketone, but iPLA2 is more effectively inhibited by a haloenol lactone suicide substrate than cPLA2. RT-PCR experiments with RNA from purified islet beta-cells and from an alpha-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the beta-cell population. Immunoblotting analyses indicate that islets express cPLA2-immunoreactive protein, and that interleukin-1 does not affect its expression. The cPLA2 is thus one of at least three classes of PLA2 enzymes with distinct properties expressed in beta-cells.     

alpha-Cell neogenesis in an animal model of IDDM

Currently there is debate regarding the capacity of pancreatic islets to regenerate in adult animals. Because pancreatic endocrine cells are thought to arise from duct cells, we examined the pancreatic ductal epithelium of the diabetic NOD mouse for evidence of islet neogenesis. We have evidence of duct proliferation as well as ductal cell differentiation, as suggested by bromodeoxyuridine-labeling and the presence of glucagon-containing cells within these ducts. In addition, the ductal epithelia in diabetic NOD mice expressed the neuroendocrine markers neuropeptide Y and tyrosine hydroxylase. These ducts also expressed the homeobox gene product, insulin promoter factor 1. Ductal cell proliferation and expression of these markers was not observed in transgenic NOD mice (NOD-E), which do not develop clinical or histopathological symptoms of IDDM. This suggests that the observed ductal cell proliferation and differentiation was a direct result of beta-cell destruction and insulin insufficiency in these adult diabetic mice, which further suggests that these events are recapitulating islet ontogeny observed during embryogenesis. It is possible that comparable processes occur in the human diabetic pancreas.     

Mouse pancreatic beta-cells exhibit preserved glucose competence after disruption of the glucagon-like peptide-1 receptor gene

Previous work suggested that glucagon-like peptide 1 (GLP-1) can acutely regulate insulin secretion in two ways, 1) by acting as an incretin, causing amplification of glucose-induced insulin release when glucose is given orally as opposed to intravenous glucose injection; and 2) by keeping the beta-cell population in a glucose-competent state. The observation that mice with homozygous disruption of the GLP-1 receptor gene are diabetic with a diminished incretin response to glucose underlines the first function in vivo. Isolated islets of Langerhans from GLP-1 receptor -/- mice were studied to assess the second function in vitro. Absence of pancreatic GLP-1 receptor function was observed in GLP-1 receptor -/- mice, as exemplified by loss of [125I]GLP-1 binding to pancreatic islets in situ and by the lack of GLP-1 potentiation of glucose-induced insulin secretion from perifused islets. Acute glucose competence of the beta-cells, assessed by perifusing islets with stepwise increases of the medium glucose concentration, was well preserved in GLP-1 receptor -/- islets in terms of insulin secretion. Furthermore, neither islet nor total pancreatic insulin content was significantly changed in the GLP-1 receptor -/- mice when compared with age-and sex-matched controls. In conclusion, mouse islets exhibit preserved insulin storage capacity and glucose-dependent insulin secretion despite the loss of functional GLP-1 receptors. The results demonstrate that the glucose responsiveness of islet beta-cells is well preserved in the absence of GLP-1 receptor signaling.     

Molecular or pharmacologic perturbation of the link between glucose and lipid metabolism is without effect on glucose-stimulated insulin secretion. A re-evaluation of the long-chain acyl-CoA hypothesis

The mechanism by which glucose stimulates insulin secretion from the pancreatic islets of Langerhans is incompletely understood. It has been suggested that malonyl-CoA plays a regulatory role by inhibiting fatty acid oxidation and promoting accumulation of cytosolic long-chain acyl-CoA (LC-CoA). In the current study, we have re-evaluated this "long-chain acyl-CoA hypothesis" by using molecular and pharmacologic methods to perturb lipid metabolism in INS-1 insulinoma cells or rat islets during glucose stimulation. First, we constructed a recombinant adenovirus containing the cDNA encoding malonyl-CoA decarboxylase (AdCMV-MCD), an enzyme that decarboxylates malonyl-CoA to acetyl-CoA. INS-1 cells treated with AdCMV-MCD had dramatically lowered intracellular malonyl CoA levels compared with AdCMV-betaGal-treated cells at both 3 and 20 mM glucose. Further, at 20 mM glucose, AdCMV-MCD-treated cells were less effective at suppressing [1-14C]palmitate oxidation and incorporated 43% less labeled palmitate and 50% less labeled glucose into cellular lipids than either AdCMV-betaGAL-treated or untreated INS-1 cells. Despite the large metabolic changes caused by expression of MCD, insulin secretion in response to glucose was unaltered relative to controls. The alternative, pharmacologic approach for perturbing lipid metabolism was to use triacsin C to inhibit long-chain acyl-CoA synthetase. This agent caused potent attenuation of palmitate oxidation and glucose or palmitate incorporation into cellular lipids and also caused a 47% decrease in total LC-CoA. Despite this, the drug had no effect on glucose-stimulated insulin secretion in islets or INS-1 cells. We conclude that significant disruption of the link between glucose and lipid metabolism does not impair glucose-stimulated insulin secretion in pancreatic islets or INS-1 cells.     

Inhibitory effects of streptozotocin, tumor necrosis factor-alpha, and interleukin-1beta on glucokinase activity in pancreatic islets and gene expression of GLUT2 and glucokinase

Treatment of streptozotocin (ST), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) resulted in destroying insulin-secreting beta-cells of pancreatic islets and impairment of islet glucose oxidation and glucose-induced insulin secretion. IL-1beta and TNF-alpha inhibited insulin release and glucose utilization and oxidation. It was shown that the inhibitory effects of ST, IL-1beta, and TNF-alpha were due to impaired glucokinase activity. Glucokinase activity was severely impaired by ST, IL-1beta, and TNF-alpha treatments, as confirmed by assaying enzymes and nucleotides associated with glycolysis and glucose oxidation. On the other hand, nitric oxide was a factor of the deleterious effects of IL-1beta, TNF-alpha, and ST on pancreatic islets. Incubation of mouse pancreatic islets with ST at various concentrations of impairing insulin secretion resulted in generation of nitrite, stimulation of islet guanylyl cyclase and accumulation of cGMP, and inhibition of pancreatic islet mitochondrial aconitase activity to degree similar to those raised by IL-1beta and TNF-alpha. When the effects of IL-1beta and TNF-alpha on the gene expression of pancreatic GLUT2 and glucokinase were examined, the level of GLUT2 and glucokinase mRNA in pancreatic islets was significantly decreased. This suggested that IL-1beta and TNF-alpha downregulate gene expression of GLUT2 and glucokinase in pancreatic beta-cells.     

IL-4-dependent regulation of TGF-alpha and TGF-beta1 expression in human eosinophils

TGFs play important roles in wound healing and carcinogenesis. We have previously demonstrated that eosinophils infiltrating into different pathologic processes elaborate TGF-alpha and TGF-beta1. Eosinophils infiltrating hamster cutaneous wounds were found to express TGFs sequentially. In this study, we examined the biologic mediators that may regulate the expression of TGF-alpha and -beta1 by eosinophils. Eosinophils were isolated from the peripheral blood of healthy donors and cultured in the absence or presence of IL-3, IL-4, and IL-5. Cells were analyzed by in situ hybridization and immunohistochemistry. Supernatants from these cultures were assayed for secreted TGF-alpha and TGF-beta1 using TGF-specific ELISAs. IL-3, IL-4, and IL-5 independently up-regulated TGF-beta1 mRNA and product expression by eosinophils in all donors. Interestingly, TGF-alpha production by eosinophils was up-regulated by IL-3 and IL-5 but was down-regulated by IL-4. Consistent with the ability of IL-4 to regulate eosinophil responses, IL-4 signaling molecules are present in human eosinophils. The observation that IL-4 can differentially regulate the expression of TGF-alpha and TGF-beta1 suggests that IL-4 may serve as a physiologic molecular switch of TGF expression by the infiltrating eosinophils in wound healing and carcinogenesis.     

Characterization of SNARE protein expression in beta cell lines and pancreatic islets

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.     

Gastric inhibitory polypeptide (GIP) and GIP receptor (GIPR)

Gastric inhibitory polypeptide, originally isolated from porcine intestine, is a gastrointestinal hormone belonging to the vasoactive intestinal peptide (VIP)/glucagon/secretin family. GIP consists of 42 amino acid residues which is derived by proteolytic processing of a GIP precursor. In vivo and in vitro experiments have indicated that GIP auguments glucose-stimulated insulin secretion, suggesting that GIP plays an important role in the regulation of insulin secretion as an incretin. Thus, GIP now is generally referred to as glucose-dependent insulinotropic polypeptide. It is also suggested that GIP may be involved in the pathogenesis of non insulin-dependent diabetes mellitus (NIDDM). GIP exerts its biological actions by binding to its specific receptors, which appear to be coupled to G proteins. We have isolated a cDNA encoding a GIP receptor from a hamster insulinoma(HIT-T15) cDNA library. The hamster GIP receptor is a 462 amino acid protein having seven transmembrane segments. Expression of recombinant of hamster GIP receptors in Chinese hamster ovary (CHO) cells shows that it binds specifically to GIP with high affinity (IC50 = 9.6 nM) and is positively coupled to adenylate cyclase. RNA blot analysis reveals that a 3.8-kb GIP receptor mRNA is expressed at high levels in rat pancreatic islets as well as in HIT-T15 cells.