Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide.

Pancreatic islet amyloid deposits are a characteristic pathologic feature of non-insulin-dependent diabetes mellitus and contain islet amyloid polypeptide (IAPP; amylin). We used transgenic mice that express human IAPP in pancreatic beta cells to explore the potential role of islet amyloid in the pathogenesis of non-insulin-dependent diabetes mellitus. Extensive amyloid deposits were observed in the pancreatic islets of approximately 80% of male transgenic mice > 13 months of age. Islet amyloid deposits were rarely observed in female transgenic mice (11%) and were never seen in nontransgenic animals. Ultrastructural analysis revealed that these deposits were composed of human IAPP-immunoreactive fibrils that accumulated between beta cells and islet capillaries. Strikingly, approximately half of the mice with islet amyloid deposits were hyperglycemic (plasma glucose > 11 mM). In younger (6- to 9-month-old) male transgenic mice, islet amyloid deposits were less commonly observed but were always associated with severe hyperglycemia (plasma glucose > 22 mM). These data indicate that expression of human IAPP in beta cells predisposes male mice to the development of islet amyloid and hyperglycemia. The frequent concordance of islet amyloid with hyperglycemia in these mice suggests an interdependence of these two conditions and supports the hypothesis that islet amyloid may play a role in the development of hyperglycemia.

Inositol 1,4,5-trisphosphate receptor subtype 3 in pancreatic islet cell secretory granules revisited.

It has been reported that the inositol 1,4,5-trisphosphate receptor subtype 3 is expressed in islet cells and is localized to both insulin and somatostatin granules [Blondel, O., Moody, M. M., Depaoli, A. M., Sharp, A. H., Ross, C. A., Swift, H. & Bell, G. I. (1994) Proc. Natl. Acad. Sci. USA 91, 7777-7781]. This subcellular localization was based on electron microscope immunocytochemistry using antibodies (affinity-purified polyclonal antiserum AB3) directed to a 15-residue peptide of rat inositol trisphosphate receptor subtype 3. We now show that these antibodies cross-react with rat, but not human, insulin. Accordingly, the anti-inositol trisphosphate receptor subtype 3 (AB3) antibodies label electron dense cores of mature (insulin-rich) granules of rat pancreatic beta cells, and rat granule labeling was blocked by preabsorption of the AB3 antibodies with rat insulin. The immunostaining of immature, Golgi-associated proinsulin-rich granules with AB3 antibodies was very weak, indicating that cross-reactivity is limited to the hormone and not its precursor. Also, the AB3 antibodies labeled pure rat insulin crystals grown in vitro but failed to stain crystals grown from pure human insulin. By immunoprecipitation, the antibodies similarly displayed a higher affinity for rat than for human insulin. We could not confirm the labeling of somatostatin granules using AB3 antibodies.

Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene.

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.

Role of heme regulated eIF2α kinase in pancreatic beta cell function and viability

The eukaryotic translation initiation factor 2 alpha (eIF2α) is part of the initiation complex that drives the initiator amino acid methionine to the ribosome, a crucial step in protein translation. In stress conditions such as virus infection, endoplasmic reticulum (ER) stress, amino acid or heme deficiency eIF2α can be phosphorylated and thereby inhibit global protein synthesis. This adaptive mechanism prevents protein accumulation and consequent cytotoxic effects. Heme-regulated eIF2α kinase (HRI) is a member of the eIF2α kinase family that regulates protein translation in heme deficiency conditions. Although present in all tissues, HRI is predominantly expressed in erythroid cells where it remains inactive in the presence of normal heme concentrations. In response to heme deficiency, HRI is activated and phosphorylates eIF2α decreasing globin synthesis. This mechanism is important to prevent accumulation of heme-free globin chains which cause ER stress and apoptosis. RNA sequencing data from our group showed that in human islets and in primary rat beta cells HRI is the most expressed eIF2α kinase compared to the other family members. Despite its high expression levels, little is known about HRI function in beta cells. The aim of this project is to identify the role of HRI in pancreatic beta cells. This was investigated taking a loss-of-function approach. HRI knock down (KD) by RNA interference induced beta cell apoptosis in basal condition. HRI KD potentiated the apoptotic effects of palmitate or proinflammatory cytokines, two in vitro models for type 2 and type 1 diabetes, respectively. Increased cytokine-induced apoptosis was also observed in HRI-deficient primary rat beta cells. Unexpectedly, we observed a mild increase in eIF2α phosphorylation in HRI-deficient cells. The levels of mRNA or protein expression of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) were not modified. HRI KD cells have decreased spliced X-box binding protein 1 (XBP1s), an important branch of the ER stress response. However, overexpression of XBP1s by adenovirus in HRI KD cells did not protect from HRI siRNA-induced apoptosis. HRI deficiency decreased phosphorylation of Akt and its downstream targets glycogen synthase kinase 3 (GSK3), forkhead box protein O1 (FOXO1) and Bcl-2-associated death promoter (BAD). Overexpression of a constitutively active form of Akt by adenovirus in HRI-deficient beta cells partially decreased HRI KD-mediated apoptosis. Interestingly, BAD silencing protected from apoptosis caused by HRI deficiency. HRI silencing in beta cells also induced JNK activation. These results suggest an important role of HRI in beta cell survival through modulation of the Akt/BAD pathway. Thus, HRI may be an interesting target to modulate beta cell fate in diabetic conditions.

Human amniotic epithelial cells induce localized cell-mediated immune privilege in vitro:implications for pancreatic islet transplantation

Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p <0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 µg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of ß-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may reduce the need for chronic systemic immunosuppression, thus making islet transplantation a more attractive treatment option for the management of insulin-dependent diabetes.

Resistin down-regulates insulin receptor expression, and modulates cell viability in rodent pancreatic beta-cells

The adipokine resistin is known to induce insulin resistance in rodent tissues. Increases in adipose tissue mass are known to have a negative effect on pancreatic beta-cell function, although the mechanisms are poorly understood. This study investigated the effects of resistin on insulin secretion, insulin receptor expression and cell viability in pancreatic beta-cells. BTC-6 or BRIN-BD11 cells were treated for 24h with resistin, and insulin receptor expression, insulin secretion and cell viability were measured. Incubation with 40ng/ml resistin caused significant decreases in insulin receptor mRNA and protein expression, but did not affect insulin secretion. At low concentrations, resistin caused significant increases in cell viability. These data implicate resistin as a factor that may regulate beta-cell function/viability, and suggests a potential mechanism by which increased adiposity causes beta-cell dysfunction.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P=0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P=0.001), sustained into adolescence (P=0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P=0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P=0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P=0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Impaired Incretin Secretion And Pancreatic Dysfunction With Older Age And Diabetes.

To estimate the impact of aging and diabetes on insulin sensitivity, beta-cell function, adipocytokines, and incretin production. Hyperglycemic clamps, arginine tests and meal tolerance tests were performed in 50 non-obese subjects to measure insulin sensitivity (IS) and insulin secretion as well as plasma levels of glucagon, GLP-1 and GIP. Patients with diabetes and healthy control subjects were divided into the following groups: middle-aged type 2 diabetes (MA-DM), aged Type 2 diabetes (A-DM) and middle-aged or aged subjects with normal glucose tolerance (MA-NGT or A-NGT). IS, as determined by the homeostasis model assessment, glucose infusion rate, and oral glucose insulin sensitivity, was reduced in the aged and DM groups compared with MA-NGT, but it was similar in the MA-DM and A-DM groups. Insulinogenic index, first and second phase insulin secretion and the disposition indices, but not insulin response to arginine, were reduced in the aged and DM groups. Postprandial glucagon production was higher in MA-DM compared to MA-NGT. Whereas the GLP-1 production was reduced in A-DM, no differences between groups were observed in GIP production. In non-obese subjects, diabetes and aging impair insulin sensitivity. Insulin production is reduced by aging, and diabetes exacerbates this condition. Aging associated defects superimposed diabetic physiopathology, particularly regarding GLP-1 production. On the other hand, the glucose-independent secretion of insulin was preserved. Knowledge of the complex relationship between aging and diabetes could support the development of physiopathological and pharmacological based therapies.

A New Goniothalamin N-acylated Aza-derivative Strongly Downregulates Mediators Of Signaling Transduction Associated With Pancreatic Cancer Aggressiveness.

In this study, a novel concise series of molecules based on the structure of goniothalamin (1) was synthesized and evaluated against a highly metastatic human pancreatic cancer cell line (Panc-1). Among them, derivative 8 displayed a low IC50 value (2.7 μM) and its concentration for decreasing colony formation was 20-fold lower than goniothalamin (1). Both compounds reduced the levels of the receptor tyrosine kinase (AXL) and cyclin D1 which are known to be overexpressed in pancreatic cancer cells. Importantly, despite the fact that goniothalamin (1) and derivative 8 caused pancreatic cancer cell cycle arrest and cell death, only derivative 8 was able to downregulate pro-survival and proliferation pathways mediated by mitogen activated protein kinase ERK1/2. Another interesting finding was that Panc-1 cells treated with derivative 8 displayed a strong decrease in the transcription factor (c-Myc), hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein levels. Notably, the molecular effects caused by derivative 8 might not be related to ROS generation, since no significant production of ROS was observed in low concentrations of this compound (from 1.5 up to 3 μM). Therefore, the downregulation of important mediators of pancreatic cancer aggressiveness by derivative 8 reveals its great potential for the development of new chemotherapeutic agents for pancreatic cancer treatment.

Chronic Continuous Exenatide Infusion Does Not Cause Pancreatic Inflammation And Ductal Hyperplasia In Non-human Primates.

In this study, we aimed to evaluate the effects of exenatide (EXE) treatment on exocrine pancreas of nonhuman primates. To this end, 52 baboons (Papio hamadryas) underwent partial pancreatectomy, followed by continuous infusion of EXE or saline (SAL) for 14 weeks. Histological analysis, immunohistochemistry, Computer Assisted Stereology Toolbox morphometry, and immunofluorescence staining were performed at baseline and after treatment. The EXE treatment did not induce pancreatitis, parenchymal or periductal inflammatory cell accumulation, ductal hyperplasia, or dysplastic lesions/pancreatic intraepithelial neoplasia. At study end, Ki-67-positive (proliferating) acinar cell number did not change, compared with baseline, in either group. Ki-67-positive ductal cells increased after EXE treatment (P = 0.04). However, the change in Ki-67-positive ductal cell number did not differ significantly between the EXE and SAL groups (P = 0.13). M-30-positive (apoptotic) acinar and ductal cell number did not change after SAL or EXE treatment. No changes in ductal density and volume were observed after EXE or SAL. Interestingly, by triple-immunofluorescence staining, we detected c-kit (a marker of cell transdifferentiation) positive ductal cells co-expressing insulin in ducts only in the EXE group at study end, suggesting that EXE may promote the differentiation of ductal cells toward a β-cell phenotype. In conclusion, 14 weeks of EXE treatment did not exert any negative effect on exocrine pancreas, by inducing either pancreatic inflammation or hyperplasia/dysplasia in nonhuman primates.

Research Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

Background: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). Patients and Methods: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGF beta 1, TGF beta 2, TGF beta 3 in oral SCC. Results: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2(-) and Smad6(+) oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2(+)/Smad6(-) subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGF beta isoforms, we found that Smad2 mRNA and TGF beta 1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGF beta 1(+) patients were Smad2(-). In larynx SCC, Smad7(-) patients did not reach mOS whereas mOS of Smad7(+) patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. Conclusion: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGF beta signaling should be better clarified in the future.

Generation and characterization of human insulin-releasing cell lines

Background: The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results: We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion: We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.