BackgroundInsulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression.Methodology/Principal FindingsGenomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro.Conclusions/SignificanceOur findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

Last year, from the approximately 6,000 organ donors, only approximately 1,500 pancreata were used for clinical transplantation. Factors that contribute to this poor pancreas use include strict donor selection criteria and the requirement for short cold-ischemia time (CIT). Numerous pancreata have not been used because of long ischemia times postprocurement. Given the oxygen-rich environment of the islets in the native pancreas, it is conceivable that islets are highly susceptible to irreversible damage following prolonged ischemia. The use of continuously oxygenated perfluorohydrocarbons (PFCs), known for their high oxygen-solubility coefficients, in a two-layer culture with standard University of Wisconsin preservation media, has extended the acceptable range CIT, and, furthermore, there has been no evidence of adverse effects from PFCs on the outcome of transplanted cells, whereas they often enhance islet cell function. The purpose of this study was to use the two-layer culture method to improve donor-organ use from marginal donors. Fifteen organs were procured using the two-layer method, and 18 without using it, from donors greater than 50 years of age. Despite nonsignificant differences in age, weight of the donors, weight of the organ and CIT, the PFC group yielded an average of twofold more islet equivalents than those harvested from the control group. As a result, from the control group, only 2 of 18 organs were used for clinical islet transplantation, whereas 8 of 15 were used from the PFC group. To this end, the two-layer method may help clinicians overcome the problem of organ underuse.

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (∆OCR glc ) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ∆OCR glc obtained at both institutions were compared. Islet specificity was determined by comparing ∆OCR glc in islet and nonislet tissue. The ∆OCR glc ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (∆OCR glc was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ∆OCR glc was statistically independent of percent beta cells, demonstrating that ∆OCR glc is governed more by islet quality than by islet composition. The ∆OCR glc , but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ∆OCR glc as a measurement of islet quality for human islet transplantation.

SUMMARY Tissue regeneration diminishes with age, concurrent with declining hormone levels including growth factors such as insulin-like growth factor-1 (IGF-1). We investigated the molecular basis for such decline in pancreatic β-cells where loss of proliferation occurs early in age, and is proposed to contribute to the pathogenesis of diabetes. We studied the regeneration capacity of β-cells in mouse model where PI3K/AKT pathway downstream of insulin/IGF-1 signaling, is upregulated by genetic deletion of Pten (phosphatase and tensin homologue deleted on chromosome 10) specifically in insulin producing cells. In this model, PTEN loss prevents the decline in proliferation capacity in aged β-cells and restores the ability of aged β-cells to respond to injury induced regeneration. Using several animal and cell models where we can manipulate PTEN expression, we found that PTEN blocks cell cycle reentry through a novel pathway leading to an increase of p16ink4a, a cell cycle inhibitor characterized for its role in cellular senescence/aging. A downregulation in p16ink4a occurs when PTEN is lost as a result of cyclin D1 induction and the activation of E2F transcription factors. The activation of E2F transcriptional factors leads to methylation of p16ink4a promoter, an event that is mediated by the upregulation of polycomb protein, Ezh2. These analyses establish a novel PTEN/cyclin D1/E2F/Ezh2/p16ink4a signaling network responsible for the aging process and provide specific evidence for a molecular paradigm that explain how decline of growth factor signals such as IGF-1 (through PTEN/PI3K signaling) may control regeneration and the lack thereof in aging cells.

Islets transplantation, as a treatment of type 1 diabetes, faces challenges, including the loss of islets in the process of isolation and pre-transplantation due to cellular stresses-induced apoptosis. Accordingly, the optimization of culture plays a decisive role in the transplantation success. In this study, we evaluated the effect of nobiletin on the cultured human islets. Isolated human islets were treated by different concentrations of nobiletin and cultured for 24 and 72 hours. Then, the islets viability, apoptosis, insulin and C-peptide secretion, and apoptosis markers were evaluated. Also, the production of reactive oxygen species (ROS), hypoxia inducible factor 1 alpha (HIF-1α), and its target genes in the islets were examined. Our findings showed that the islets were encountered with hypoxia and oxidative stress after isolation and during culture. These insults induced apoptosis and reduced viability during culture period. Moreover, the secretion of insulin and C-peptide decreased. Nobiletin treatments significantly improved the islets survival through reduction of HIF-1α and ROS production and suppression of apoptosis, along with increased islets function. Islet protective effect of nobiletin might be related to its anti-oxidant, anti-apoptotic and insulinotropic properties. Hence, in order to achieve viable and functional islets for clinical transplantation, the application of nobiletin during pre-transplantation period is useful.