Insulin acts as a repressive factor to inhibit the ability of PAR2 to induce islet cell transdifferentiation.

Recently, we showed that pancreatitis in the context of profound ß-cell deficiency was sufficient to induce islet cell transdifferentiation. In some circumstances, this effect was sufficient to result in recovery from severe diabetes. More recently, we showed that the molecular mechanism by which pancreatitis induced ß-cell neogenesis by transdifferentiation was activation of an atypical GPCR called Protease-Activated Receptor 2 (PAR2). However, the ability of PAR2 to induce transdifferentiation occurred only in the setting of profound ß-cell deficiency, implying the existence of a repressive factor from those cells. Here we show that the repressor from ß-cells is insulin. Treatment of primary islets with a PAR2 agonist (2fLI) in combination with inhibitors of insulin secretion and signaling was sufficient to induce insulin and PAX4 gene expression. Moreover, in primary human islets, this treatment also led to the induction of bihormonal islet cells coexpressing glucagon and insulin, a hallmark of islet cell transdifferentiation. Mechanistically, insulin inhibited the positive effect of a PAR2 agonist on insulin gene expression and also led to an increase in PAX4, which plays an important role in islet cell transdifferentiation. The studies presented here demonstrate that insulin represses transdifferentiation of α- to ß-cells induced by activation of PAR2. This provides a mechanistic explanation for the observation that α- to ß-cell transdifferentiation occurs only in the setting of severe ß-cell ablation. The mechanistic understanding of islet cell transdifferentiation and the ability to modulate that process using available pharmacological reagents represents an important step along the path towards harnessing this novel mechanism of ß-cell neogenesis as a therapy for diabetes.

Executive Summary of IPITA-TTS Opinion Leaders Report on the Future of ß-Cell Replacement.

The International Pancreas and Islet Transplant Association (IPITA), in conjunction with the Transplantation Society (TTS), convened a workshop to consider the future of pancreas and islet transplantation in the context of potential competing technologies that are under development, including the artificial pancreas, transplantation tolerance, xenotransplantation, encapsulation, stem cell derived beta cells, beta cell proliferation, and endogenous regeneration. Separate workgroups for each topic and then the collective group reviewed the state of the art, hurdles to application, and proposed research agenda for each therapy that would allow widespread application. Herein we present the executive summary of this workshop that focuses on obstacles to application and the research agenda to overcome them; the full length article with detailed background for each topic is published as an online supplement to Transplantation.

Simultaneous silencing of multiple RB and p53 pathway members induces cell cycle reentry in intact human pancreatic islets.

BACKGROUND: Human pancreatic islet structure poses challenges to investigations that require specific modulation of gene expression. Yet dissociation of islets into individual cells destroys cellular interactions important to islet physiology. Approaches that improve transient targeting of gene expression in intact human islets are needed in order to effectively perturb intracellular pathways to achieve biological effects in the most relevant tissue contexts. RESULTS: Electroporation of intact human cadaveric islets resulted in robust and specific suppression of gene expression. Two genes were simultaneously suppressed by 80% from baseline levels. When multiple (up to 5) genes were simultaneously targeted, effective suppression of 3 of 5 genes occurred. Enzymatic pretreatment of islets was not required. Simultaneous targeting of RB and p53 pathway members resulted in cell cycle reentry as measured by EDU incorporation in 10% of islet nuclei. CONCLUSIONS: At least three genes can be effectively suppressed simultaneously in cultured intact human pancreatic islets without disruption of islet architecture or overt alterations in function. This enabled the effective modulation of two central growth control pathways resulting in the phenotypic outcome of cell cycle reentry in postmitotic islet cells. Transient exposure to multiple siRNAs is an effective approach to modify islets for study with the potential to aid clinical applications.

Encapsulated islets for diabetes therapy: history, current progress, and critical issues requiring solution.

Insulin therapy became a reality in 1921 dramatically saving lives of people with diabetes, but not protecting them from long-term complications. Clinically successful free islet implants began in 1989 but require life long immunosuppression. Several encapsulated islet approaches have been ongoing for over 30 years without defining a clinically relevant product. Macro-devices encapsulating islet mass in a single device have shown long-term success in large animals but human trials have been limited by critical challenges. Micro-capsules using alginate or similar hydrogels encapsulate individual islets with many hundreds of promising rodent results published, but a low incidence of successful translation to large animal and human results. Reduction of encapsulated islet mass for clinical transplantation is in progress. This review covers the status of both early and current studies including the presentation of corporate efforts involved. It concludes by defining the critical items requiring solution to enable a successful clinical diabetes therapy.

Pulsatile intravenous insulin therapy: the best practice to reverse diabetes complications?

In the basal state and after oral ingestion of carbohydrate, the normal pancreas secretes insulin into the portal vein in a pulsatile manner. The end organ of the portal vein is the liver, where approximately 80% of pancreatic insulin is extracted during first pass. In Type 1 diabetes, pancreatic insulin secretion is nearly or completely absent whilst in Type 2 diabetes the normal pattern is absent, abnormal, or blunted. Exogenous subcutaneous insulin treatment results in plasma insulin concentrations that are not pulsatile and a fraction of normal portal vein levels. Oral hypoglycemic agents also do not result in normal pulsatile response to a glucose load. Due to hypoglycemia risk, intensive treatment is not recommended after serious complications develop. Consequently, no conventional therapy has proved effective in treating advanced diabetes complications. Beta-cell replacement using whole pancreas or islet transplantation has been utilized to treat certain problems in Type 1 diabetic patients, but still unavailable for all diabetics. Pulsatile intravenous insulin therapy (PIVIT) is an insulin therapy, which mimics the periodicity and amplitude of normal pancreatic function. Numerous studies show PIVIT effective in preventing, reversing, and reducing the severity and progression of diabetes complications, however, the mechanisms involved with the improvement are not clearly understood. Here, we review the cellular basis of normal and abnormal insulin secretion, current treatments available to treat diabetes, the physiologic basis of PIVIT and possible mechanisms of action.

Hypothermic storage and preservation of human pancreatic acinar tissue.

Pancreatic acinar cells rapidly lose their characteristic features when cultured in vitro. No successful cryopreservation methods have been reported. To solve the problem of storing pancreatic acinar material, we found that it could be preserved at nonfreezing, cold temperatures: above the freezing point of cell culture medium (-0.6 degrees C) or at typical refrigeration temperatures (6.0-8.0 degrees C) for up to 7 d. Under the conditions we defined, we determined that there was no significant dedifferentiation and no significant decrease in cell health. Good viability and enzyme content were realized after storage, as determined by growth in culture, histological evaluation, and enzyme content by ELISA (lipase and amylase).