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Pro-inflammatory cytokines induce death of beta cells and hamper engraftment of transplanted islet mass. Our aim was to reveal novel genes involved in this process, as a platform for innovative therapeutic approaches.Small interfering RNA (siRNA) high-throughput screening (HTS) of primary human islets was employed to identify novel genes involved in cytokine-induced beta cell apoptosis. Dispersed human islets from nine human donors, treated with a combination of TNF-α, IL-1β and IFN-γ were transfected with ∼730 different siRNAs. Caspase-3/7 activity was measured, results were analysed and potential anti- and pro-apoptotic genes were identified.Dispersed human pancreatic islets appeared to be suitable targets for performance of siRNA HTS. Using this methodology we found a number of potential pro- and anti-apoptotic target hits that have not been previously associated with pancreatic beta cell death. One such hit was the de-ubiquitinating enzyme otubain 2 (OTUB2). OTUB2 knockdown increased caspase-3/7 activity in MIN6 cells and primary human islets and inhibited insulin secretion and increased nuclear factor-κB (NF-κB) activity both under basal conditions and following cytokine treatment.Use of dispersed human islets provides a new platform for functional HTS in a highly physiological system. Employing this technique enabled the identification of OTUB2 as a novel promoter of viability and insulin secretion in human beta cells. OTUB2 acts through the inhibition of NF-κB signalling, which is deleterious to beta cell survival. siRNA screens of human islets may therefore identify new targets, such as OTUB2, for therapeutic intervention in type 1 diabetes and islet transplantation.

Diabetic neuropathy is a common complication of diabetes that is often associated both with considerable morbidity and mortality. The epidemiology and natural course of diabetic neuropathy is clouded with uncertainty, largely due to confusion regarding the definition and measurement of this disorder. The recent resurgence of interest in the vascular hypothesis, oxidative stress, the neurotrophic hypothesis and the possibility of the role of autoimmunity have opened up new avenues of investigation for therapeutic intervention. Paralleling our increased understanding of the pathogenesis of diabetic neuropathy, there must be refinements in our ability to measure quantitatively the different types of defects that occur in this disorder, so that appropriate therapies can be targeted to specific fibre types. These tests must be validated and standardised to allow comparability between studies and a more meaningful interpretation of study results. Our ability to manage successfully the many different manifestations of diabetic neuropathy depends ultimately on our success in uncovering the pathogenic processes underlying this disorder.

Partial pancreatic duct obstruction in the hamster leads to the induction of endocrine-cell differentiation and new islet formation. We prepared cytosolic extracts from the partially obstructed pancreas and identified one, which when administered i.p., produced significant increases in the incorporation of tritiated thymidine by ductular and islet cells, as well as a corresponding increase in islet mass. In this study, we evaluate the ability of this extract to reverse streptozotocin diabetes mellitus. Hamsters were treated i.p. twice daily for 7 weeks with either 0.9% NaCl (saline) (n = 10) or a cytosol extract (n = 10) prepared previously from partially obstructed hamster pancreata. All animals in the cytosol group survived vs only 60% of the saline group (p = 0.02). Random blood glucose levels were greater than 22.2 mmol/l in 90% of the saline group vs 40% in the cytosol group (p < 0.05). Pancreatic tissue from the surviving saline animals and from persistently hyperglycaemic cytosol-treated animals, showed intra-cytoplasmic vacuolation of islet cells, a characteristic lesion of sustained hyperglycaemic states. Vacuolation was not observed in normoglycaemic extract treated animals. Islets in hyperglycaemic animals demonstrated a profound decrease or absence of immunoreactive insulin, compared to an abundance of immunoreactive beta cells in cytosol-treated animals that reverted to normoglycaemia. In this group, single cells or nests of cells stained for insulin on glucagon cells were identified in ductal epithelium in association with cells budding from the duct. Morphometric analysis of pancreata in reverted cytosol-treated animals showed a new population of small islets compared with saline controls and an increased islet mass. In summary, streptozotocin diabetes can be reversed by new islet formation induced by local pancreatic growth factors, the exact nature of which remains to be determined.

Endogenous insulin deficiency is the tie that binds all forms of diabetes. Definitive treatment or cure of type 1 and type 2 diabetes cannot occur without a durable reversal of relative or absolute endogenous insulin deficiency. Current efforts to restore physiological insulin secretion include (1) islet or pancreas transplant; (2) exogenous cell therapies, including differentiation of bone marrow cells and embryonic stem cells (ESCs); and (3) islet regeneration from endogenous pancreatic cells. Although they would represent advances, islet or beta cell transplant [1, 2], the artificial pancreas [3, 4], gene therapy [5], and even stem cell therapy [6, 7] cannot be considered restorative approaches unless the results are durable and have an acceptable safety profile. Islet transplantation, both alloand xeno-, as a means of reversing diabetes has been limited by graft durability and/ or donor supply, as well as the complications of surgery and immunosuppression. However, progress is being made to resolve these issues [1, 8]. Minimally invasive techniques for the transplant of donor islets into the liver have been developed, and extensive studies have refined the immunosuppression required for transplant survival. At present, the limiting problem for islet transplantation is obtaining sufficient islets for the broad application of the technique. It is hoped that work with stem cells will resolve this issue. Early studies showed that ESCs did not achieve terminal differentiation and had a low insulin content and a poor response to glucose. Growth of the islet-like clusters from ESCs was unbridled and, despite production of insulin, they failed to cure diabetic mice [9]. Israeli scientists found insulin-producing cells in embryoid bodies formed spontaneously from ESCs when they stop growing, but these did not make sufficient insulin to reverse diabetes [10]. Soria and colleagues used gene-trapping techniques to isolate insulin-producing cells and transfected an antibiotic resistance gene adjoined to the insulin promoter. When these cells formed three-dimensional structures, the cells increased insulin production to therapeutic levels but insulin secretion remained insensitive to glucose [11]. More recent Diabetologia (2009) 52:735–738 DOI 10.1007/s00125-009-1322-y

We have reported previously that cellophane wrapping of the hamster pancreas is a stimulus that leads to the induction of duct epithelial cell proliferation, followed by endocrine cell differentiation and new islet formation. Reg is a candidate gene that has been reported to be expressed in regenerating pancreatic islets, suggesting a role in islet growth. We examined Reg gene expression in the cellophane-wrap model by isolating total RNA from hamster pancreata at various times after wrapping. Northern blot analysis using a rat cDNA Reg probe showed no expression of Reg in control non-wrapped hamster pancreas, whereas a strong signal was detected in control wrapped rat pancreas. Using reverse transcription of RNA followed by polymerase chain reaction (PCR) we amplified, isolated and sequenced a 194 base pair product which showed homology to rat Reg in both control and wrapped hamster pancreas. When the PCR product was used as a probe for Northern blot analysis, no signal was detected in control non-wrapped pancreata. In contrast, a strong signal was detected 1 and 2 days after wrapping, which then returned to basal between 4 and 6 days after wrapping. A similar temporal pattern was observed using in situ hybridization to localize the Reg gene. One- and 2-day wrapped but not control pancreas expressed Reg in acinar cells, but not in islets. In conclusion, (a) Reg expression is low in the control hamster pancreas; (b) in the cellophane-wrap model of islet neogenesis, increased Reg mRNA is found within acinar tissue; (c) Reg gene may thus be involved as an acinar paracrine effector of duct cell proliferation in the initial step of islet neogenesis, but may also participate in the inflammatory response to traumatic stimuli.

The relationship between insulin responses to oral glucose and pancreatic exocrine function were examined in 15 patients with chronic pancreatitis. Good correlations were found between the insulin responses and exocrine pancreatic function measured as the concentrations of pancreatic enzymes in duodenal juice after intravenous cholecystokinin-pancreazymin (CCK-PZ). There appears to be a roughly parallel loss of endocrine and exocrine function in the course of chronic pancreatitis.

This study compares blood glucose, plasma insulin and tolbutamide values throughout 48 h in 10 subjects on a single daily dose of tolbutamide and on a divided dose regimen (the two regimens are equally effective in their blood sugar lowering effect at all times throughout the 48 hours). — A plasma tolbutamide of 2–5 mg per cent was found to have some blood sugar lowering effect, and 24 h after a single oral dose effective levels were detectable in the plasma of some subjects. — Plasma insulin appears to respond to fluctuating glucose levels rather than to the tolbutamide levels and cannot fully account for the prolonged action of tolbutamide. — Tolbutamide given intravenously 3 h after a previous oral dose elicited a further rise in plasma insulin. The response was enhanced when the interval was increased to 18 h. A second peak in plasma insulin levels was often seen one hour after intravenous tolbutamide. — No insulin response was seen to repeated oral doses given at six to eight hour intervals. — An unexpected rise in plasma FFA was frequently observed ten minutes after intravenous tolbutamide. This may be attributed to the high circulating levels of tolbutamide which are measured as FFA by the method employed. — The mechanism of prolonged tolbutamide action remains unclear. These findings and their possible relationship to this action are discussed.