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.

Tumor suppressor Pdcd4 is a major transcript that is upregulated during in vivo pancreatic islet neogenesis and is expressed in both beta-cell and ductal cell lines.

OBJECTIVES: We wished to identify a major transcript that is upregulated during in vivo pancreatic islet neogenesis and examine the expression of the gene in beta and ductal cells. METHODS: Differential display polymerase chain reaction was used to identify upregulated transcripts after islet neogenesis was stimulated in the rat by brief occlusion of the main pancreatic duct. The expression of this major transcript, namely PDCD4 (programmed cell death gene 4), was measured in beta and ductal cells after stimulation with the incretin hormone glucagon-like peptide 1, mitogenic insulin, the thiazolidinedione rosiglitazone, and by high glucose concentrations. The subcellular location of the protein was also examined. RESULTS: The expression of the Pdcd4 gene in pancreatic beta and ductal cells was found to be stimulated in a comparable manner by either glucagon-like peptide 1, insulin, and by high glucose concentrations. However, intracellular localisation of the PDCD4 protein was shown to be differentially regulated by these stimuli in beta and ductal cells. Furthermore, the thiazolidinedione rosiglitazone specifically upregulates Pdcd4 gene expression in beta cells in a time-dependent manner. CONCLUSION: This is the first study showing Pdcd4 expression in pancreatic cells. Our data indicate that Pdcd4 expression may be integral in the function of the adult pancreas.

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.

Selenium stimulates pancreatic beta-cell gene expression and enhances islet function.

The present study investigated the role of selenium in the regulation of pancreatic beta-cell function. Utilising the mouse beta-cell line Min6, we have shown that selenium specifically upregulates Ipf1 (insulin promoter factor 1) gene expression, activating the -2715 to -1960 section of the Ipf1 gene promoter. Selenium increased both Ipf1 and insulin mRNA levels in Min6 cells and stimulated increases in insulin content and insulin secretion in isolated primary rat islets of Langerhans. These data are the first to implicate selenium in the regulation of specific beta-cell target genes and suggest that selenium potentially promotes an overall improvement in islet function.