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Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with (31)P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by (13)C NMR isotopomer analysis of the fate of [U-(13)C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7 mM), inflammatory cytokine addition (IL-1beta, TNF-alpha, and IFN-gamma), or both, for 48 h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and beta cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.

Confluent monolayer cultures of betaTC3 cells were exposed for 4 h to acidic, neutral, or alkaline pH media. Studies determined the impact of pH on viability, insulin secretion rate, glucose consumption rate, lactate production rate, and ATP content. Cell viability was not affected by exposure to media of different pH (>95% for all groups). Insulin release from cells exposed to acidic media (pH of 6.4) was approximately 75% higher than that from cells exposed to either neutral (pH of 7.1) or alkaline (pH of 7.8) conditions. Conversely, ATP content was significantly reduced in cultures exposed to acidic conditions, although there was no statistical difference between neutral and alkaline conditions. Glucose consumption and lactate production rates increased linearly with increasing pH., (Copyright 2000 Academic Press.)

Oxygen consumption rates of beta TC3 mouse insulinoma cells were measured in three different cell culture systems: monolayer, freshly trypsinized cell suspension, and trypsinized cells entrapped in alginate-poly-L-lysine-alginate (APA) polymer beads. The oxygen consumption rate for cells in the APA beads was similar to the rate for freshly trypsinized cells, indicating that cells can be entrapped without affecting their oxygen consumption rate. The cells in monolayers consumed oxygen at a higher rate than freshly trypsinized cells or cells in APA beads, suggesting that trypsinization may lower oxygen consumption capability. In addition, the oxygen consumption of beta TC3s during glucose-induced insulin secretion was not significantly different from the basal rate, in contrast to the increased oxygen demand of actively secreting islets. Our results suggest that beta TC3s may be better suited than islets for use in a bioartificial pancreas due to their stable oxygen requirements.