There is a need for simple, quantitative and prospective assays for islet quality assessment that are predictive of islet transplantation outcome. The current state-of-the-art athymic nude mouse bioassay is costly, technically challenging and retrospective. In this study, we report on the ability of 2 parameters characterizing human islet quality: (1) oxygen consumption rate (OCR), a measure of viable volume; and (2) OCR/DNA, a measure of fractional viability, to predict diabetes reversal in nude mice. Results demonstrate that the probability for diabetes reversal increases as the graft's OCR/DNA and total OCR increase. For a given transplanted OCR dose, diabetes reversal is strongly dependent on OCR/DNA. The OCR and OCR/DNA (the 'OCR test') data exhibit 89% sensitivity and 77% specificity in predicting diabetes reversal in nude mice (n = 86). We conclude that the prospective OCR test can effectively replace the retrospective athymic nude mouse bioassay in assessing human islet quality prior to islet transplantation.

Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic ␤-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13 C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was ϳ40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mM stimulated insulin secretion ϳ4-fold, and was associated with a ϳ4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux ϳ6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in ␤-cells and that anaplerosis through GDH does not play a major role in this process.Glucose homeostasis depends on the appropriate release of insulin from the pancreatic ␤-cell in response to increases in blood glucose concentrations. Although, it is well established that glucose-stimulated insulin secretion requires mitochondrial carbohydrate oxidation to drive ATP production (1), a number of studies have suggested that intermediates (e.g. malonyl-CoA (2, 3), succinate (4), and glutamate (5)) generated, directly or indirectly, from anaplerosis may act as second messengers linking glucose oxidation with insulin secretion.Previous studies of anaplerotic flux have typically examined only a single anaplerotic pathway (pyruvate carboxylase (PC), 1 or glutamate dehydrogenase (GDH)) with a single substrate. Given the complexity and interdependence of these fluxes, a comprehensive and simultaneous evaluation of the relative contribution and regulation of alternate anaplerotic pathways and substrates into the Kreb's cycle is essential to understand the relative role of these individual fluxes in the regulation of glucose-stimulated insulin secretion. The experimental design of most previous studies have either inhibited competing anaplerotic pathways or used methodology that preclude any analysis of the coordination of flux through competing anaplerotic pathways. To address these limitations, we examined the relationship between insulin secretion and the pathways of anaplerosis in clonal INS-1 832/13 cells under conditions of low (2.5 mM) and high (15 mM) glucose. In addition, we also examined the effec...

Purpose of review-There is a critical need for meaningful viability and potency assays that characterize islet preparations for release prior to clinical islet cell transplantation (ICT). Development, testing, and validation of such assays have been the subject of intense investigation for the past decade. These efforts are reviewed, highlighting the most recent results while focusing on the most promising assays.Recent Findings-Assays based on membrane integrity do not reflect true viability when applied to either intact islets or dispersed islet cells. Assays requiring disaggregation of intact islets into individual cells for assessment introduce additional problems of cell damage and loss. Assays evaluating mitochondrial function, specifically mitochondrial membrane potential, bioenergetic status, and cellular oxygen consumption rate (OCR), especially when conducted with intact islets, appear most promising in evaluating their quality prior to ICT. Prospective, quantitative assays based on measurements of OCR with intact islets have been developed, validated and their results correlated with transplant outcomes in the diabetic nude mouse bioassay.Conclusion-More sensitive and reliable islet viability and potency tests have been recently developed and tested. Those evaluating mitochondrial function are most promising, correlate with transplant outcomes in mice, and are currently being evaluated in the clinical setting.

We sought to determine the long-term outcomes in type 1 diabetic recipients of intraportal alloislet transplants on a modified immunosuppressive protocol. Six recipients with hypoglycemia unawareness received 1 to 2 islet infusions. Induction therapy was with antithymocyte globulin (ATG) plus etanercept for tumor necrosis factor-α blockade. Recipients received cyclosporine and everolimus for maintenance immunosuppression for the first year posttransplant, with mycophenolic acid or mycophenolate mofetil subsequently substituted for everolimus. Recipients have been followed for 1173 ± 270 days since their last infusion for islet graft function (insulin independence, hemoglobin A1c levels, and C-peptide production) and for adverse events associated with the study protocol. Of the 6 recipients, 5 were insulin-independent at 1 year, and 4 continue to be insulin-independent at a mean of 3.4 ± 0.4 years posttransplant. None of the 6 recipients experienced recurrence of severe hypoglycemia. Measured glomerular filtration rate decreased from 110.5 ±21.2 mL/min/1.73m2 pretransplant to 82.6 ±19.1 mL/min/1.73m2 at 1 year posttransplant. In conclusion, islet transplants restored insulin independence for a mean of >3 years in 4 of 6 recipients treated with ATG and etanercept induction therapy and with cyclosporine and, initially, everolimus for maintenance. Our results suggest this immunosuppressive protocol may allow long-term graft survival.