The role of tumour suppressor PDCD4 in beta cell death in hypoxia.

OBJECTIVE: Hypoxia is known to induce pancreatic beta cell dysfunction and apoptosis. Changes in Programmed Cell Death Gene 4 (PDCD4) expression have previously been linked with beta cell neogenesis and function. Our aim was to investigate the effects of hypoxia on cell viability, PDCD4 expression and subcellular localisation. METHODS: MIN6 beta cells and ARIP ductal cells were exposed to 1% (hypoxia) or 21% O2 (normoxia) for 12 or 24 hours. MTT assay, HPI staining, scanning electron microscopy, western blotting and immunocytochemistry analyses were performed to determine the effect of hypoxia on cell viability, morphology and PDCD4 expression. RESULTS: 24 hour exposure to hypoxia resulted in ~70% loss of beta cell viability (P<0.001) compared to normoxia. Both HPI staining and SEM analysis demonstrated beta cell apoptosis and necrosis after 12 hours exposure to hypoxia. ARIP cells also displayed hypoxia-induced apoptosis and altered surface morphology after 24 hours, but no significant growth difference (p>0.05) was observed between hypoxic and normoxic conditions. Significantly higher expression of PDCD4 was observed in both beta cells (P<0.001) and ductal (P<0.01) cells under hypoxic conditions compared to controls. PDCD4 expression was localised to the cytoplasm of both beta cells and ductal cells, with no observed effects of hypoxia, normoxia or serum free conditions on intracellular shuttling of PDCD4. CONCLUSION: These findings indicate that hypoxia-induced expression of PDCD4 is associated with increased beta cell death and suggests that PDCD4 may be an important factor in regulating beta cell survival during hypoxic stress.

Carboxybetaine-modified succinylated chitosan-based beads encourage pancreatic ß-cells (Min-6) to form islet-like spheroids under in vitro conditions.

In vitro, pancreatic ß-cells tend to reduce their ability to aggregate into islets and lose insulin-producing ability, likely due to insufficient cell-cell and cell-matrix interactions that are essential for ß-cell retention, viability and functionality. In response to these needs, surfaces of succinylated chitosan-based beads (NSC) were modified with zwitterionic carboxy-betaine (CB) moieties, a compatible osmolyte known to regulate cellular hydration state, and used to promote the formation of ß-cell spheroids using a conventional 2D cell culture technique. The NSC were synthesised by ionic gelation and surface-functionalised with CB using carbodiimide chemistry. Scanning electron microscopy (SEM), dynamic laser scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) were employed as characterisation tools to confirm the successful modification of the succinylated chitosan material into spherical beads with rough surfaces and a diameter of 0.4 µm. NSC with and without CB were re-suspended at concentrations of 0.1, 0.3 and 0.6 mg/mL in saline medium and tested in vitro with MIN6 murine pancreatic ß-cell line. Results showed that a concentration of 0.3 mg/mL, NSC-CB encouraged pancreatic MIN6 cells to proliferate and form spheroids via E-cadherin and Pdx-1 activation within 48 h in culture. These spheroids, with a size of approximately 80 µm, exhibited high cell viability and enhanced insulin protein expression and secretion when compared to cells organised by the non-modified beads.

Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas.

AIMS/HYPOTHESIS: Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. METHODS: Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. RESULTS: Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. CONCLUSIONS/INTERPRETATION: When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.

Immunopathology of the human pancreas in type-I diabetes.

Type 1 diabetes is a chronic autoimmune disease characterised by the selective destruction of pancreatic beta (ß) cells. The understanding of the aetiology of this disease has increased dramatically in recent years by the study of tissue recovered from patients, from analysis of the responses of isolated islet and ß-cells in tissue culture and via the use of animal models. However, knowledge of the immunopathology of type 1 diabetes in humans is still relatively deficient due largely to the difficulty of accessing appropriate samples. Here we review the state of current knowledge in relation to the histopathological features of the disease in humans. We focus specifically on recent-onset type 1 diabetes cases since in such patients, evidence of the ongoing disease process is still present. We chart the progression of the disease by describing the characteristic features of the pancreas, consider the sequence of immune cell infiltration and discuss the abnormalities of MHC antigen expression. The possibility that these changes might derive from a persistent enteroviral infection of the islet beta cells is examined.

Pseudoislets as primary islet replacements for research: report on a symposium at King's College London, London UK.

Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying ß-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 ß-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed 'pseudoislets' largely recapitulates the function of primary islet ß-cells. The Diabetes Research Group at King's College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on "Pseudoislets as primary islet replacements for research", which was funded by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or ß-cell lines but who do not currently use pseudoislets. This short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research.

Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue.

BACKGROUND: The detection of viral infection in paraffin-embedded, formalin-fixed tissue is notoriously difficult and often requires inherent knowledge about the specific virus being sought. For this reason, there is an ongoing need for reagents and methods which can identify a range of different virus types in paraffin embedded tissue. OBJECTIVES: The aim of this study was to optimise and validate the use of antisera directed against dsRNA (>50 bp in length) in paraffin-embedded formalin-fixed tissue samples. STUDY DESIGN: dsRNA antisera were optimised for use in a range of virally-infected tissue culture cells, Coxsackie-infected mice and human tissues. The specificity of labelling was confirmed by pre-adsorption of antisera with poly-IC and by digestion of dsRNA with RNaseIII. RESULTS: Two different polyclonal dsRNA antisera (J2 and K1) were capable of recognising dsRNA encoded by all the multiple different viral types (including (+) ssRNA viruses, dsRNA viruses and DNA viruses) tested in paraffin-embedded formalin fixed infected cells and tissues. In contrast, the enteroviral vp1 antisera detected only a subset of the (+) ssRNA viruses tested. Staining was not seen in uninfected cells or in uninfected control tissues. Positive staining was ablated following incubation of antisera with poly-IC or by pre-treating sections with RNaseIII prior to staining. CONCLUSIONS: The dsRNA antisera J2 and K1 are useful for the detection of viral infection in formalin-fixed, paraffin-embedded, human tissue samples.

Monitoring exercise-induced changes in glycemic control in type 2 diabetes.

PURPOSE: The present study determined the efficacy of the Continuous Glucose Monitoring System (CGMS) during moderate exercise and monitored the changes in whole-day glucose profiles using the CGMS in individuals with and without type 2 diabetes. METHODS: Six, obese, diet-treated individuals with and four age-matched individuals without type 2 diabetes were monitored using the CGMS for 3 d. Subjects cycled at 90% of a predetermined lactate threshold for 1 h at approximately 09:00 h on day 2, during which venous blood was sampled at 10-min intervals and immediately analyzed for glucose concentrations. RESULTS: Venous blood glucose and CGMS values declined during exercise in the diabetes (P < 0.001) but not the control group (P = 0.085). The CGMS overestimated blood glucose in the control (P = 0.003) and the diabetes (P = 0.045) groups during exercise. The number of data points outside of the 95% confidence intervals was <5% in both groups, showing that there is a statistically acceptable level of agreement between venous blood glucose and CGMS values during exercise. Moderate exercise improved whole-day average glucose concentrations (P = 0.007) and whole-day area under the glucose curve (P = 0.016) values (AUCglu), and the time spent within +/-10% of fasting venous glucose (FVG) increased in the diabetes group (P = 0.021). No such effects were seen in the control group. CONCLUSION: Using continuous glucose monitoring we were able to demonstrate that a period of moderate exercise improved whole-day glycemic control in obese individuals with type 2 diabetes. The CGMS should only be used as an adjunct and not as an alternative to frequent blood glucose sampling when examining the changes in glucose values during exercise in individuals with and without type 2 diabetes.

Cell-to-cell contact influences proliferative marker expression and apoptosis in MIN6 cells grown in islet-like structures.

Cell-to-cell interactions play an important role in the development and maintenance of the beta-cell phenotype. Here, we have investigated whether E-cadherin plays a role in regulating the growth of insulin-secreting MIN6 cells configured as three-dimensional islet-like clusters (pseudoislets). Pseudoislets form by cell aggregation rather than by proliferation from individual cells and attain the size of primary mouse islets after approximately 7 days of maintenance in culture. E-cadherin is known to mediate homotypic cell adhesion between beta-cells and has also been implicated in a number of cellular processes, including proliferation, apoptosis, and differentiation. E-cadherin and its associated intracellular elements, alpha- and beta-catenin, were upregulated in MIN6 pseudoislets. Pseudoislet formation was associated with an increased expression of cyclin-dependent kinase inhibitors and a concomitant downregulation of Ki67, suggesting an overall reduction in cellular proliferation. However, measurements of 5-bromo-2'-deoxyuridine incorporation revealed that there were no differences in the rate of MIN6 cell proliferation whether they were configured as monolayers or as pseudoislets, which is likely to be a result of their being a transformed cell line. Cells within pseudoislets were not necrotic, but apoptosis appeared to be upregulated in the islet-like structures. However, no differential expression of Fas and FasL was detected in monolayers and pseudoislets. These results suggest that cell-to-cell interactions within islet-like structures may initiate antiproliferative and proapoptotic signals.

Repair of cytokine-induced DNA damage in cultured rat islets of Langerhans.

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1beta (IL-1beta), interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during,the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-beta-D-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.

Antioxidant enzyme activity and mRNA expression in the islets of Langerhans from the BB/S rat model of type 1 diabetes and an insulin-producing cell line.

It has been proposed that low activities of antioxidant enzymes in pancreatic beta cells may increase their susceptibility to autoimmune attack. We have therefore used the spontaneously diabetic BB/S rat model of type 1 diabetes to compare islet catalase and superoxide dismutase activities in diabetes-prone and diabetes-resistant animals. In parallel studies, we employed the RINm5F beta cell line as a model system (previously validated) to investigate whether regulation of antioxidant enzyme activity by inflammatory mediators (cytokines, nitric oxide) occurs at the gene or protein expression level. Diabetes-prone rat islets had high insulin content at the age used (58-65 days) but showed increased amounts of DNA damage when subjected to cytokine or hydrogen peroxide treatments. There was clear evidence of oxidative damage in freshly isolated rat islets from diabetes-prone animals and significantly lower catalase and superoxide dismutase activities than in islets from age-matched diabetes-resistant BB/S and control Wistar rats. The mRNA expression of antioxidant enzymes in islets from diabetes-prone and diabetes-resistant BB/S rats and in RINm5F cells, treated with a combination of cytokines or a nitric oxide donor, DETA-NO, was analysed semi-quantitatively by real time PCR. The mRNA expression of catalase was lower, whereas MnSOD expression was higher, in diabetes-prone compared to diabetes-resistant BB/S rat islets, suggesting regulation at the level of gene expression as well as of the activities of these enzymes in diabetes. The protein expression of catalase, CuZnSOD and MnSOD was assessed by Western blotting and found to be unchanged in DETA-NO treated cells. Protein expression of MnSOD was increased by cytokines in RINm5F cells whereas the expression of CuZnSOD was slightly decreased and the level of catalase protein was unchanged. We conclude that there are some changes, mostly upregulation, in protein expression but no decreases in the mRNA expression of catalase, CuZnSOD or MnSOD enzymes in beta cells treated with either cytokines or DETA-NO. The lower antioxidant enzyme activities observed in islets from diabetes-prone BB/S rats could be a factor in the development of disease and in susceptibility to DNA damage in vitro and could reflect islet alterations prior to immune attack or inherent differences in the islets of diabetes-prone animals, but are not likely to result from cytokine or nitric oxide exposure in vivo at that stage.