Development and Application of Analytical Techniques for Evaluating Function in Pancreatic Islets of Langerhans.

Abstract

Type 1 diabetes is caused by autoimmune destruction of insulin-secreting beta-cells found in the islets of Langerhans of the pancreas. Severe cases can be treated in a minimally invasive way by islet transplantation; however, islet transplantation has been limited by an inability to measure islet viability and potency prior to transplant. To address this need, we have developed a microfluidic platform to measure both intracellular calcium flux and insulin secretion, two important indicators of beta-cell function, at high temporal resolution during glucose treatment. Combining these measures on islets required methods for measuring fluorescence at two separate locations on a microfluidic system. To accomplish this objective, we used a 2-chip system in which perfusate was collected in fractions while intracellular calcium was measured using fluorescence imaging. The perfusate was subsequently analyzed for insulin by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) using the same fluorescence microscope. We were able to distinguish first and second phase insulin secretion from batches of 8-10 islets with 80 s temporal resolution. Measured basal and peak first phase insulin secretion correlated well with previously reported results. Total analysis time using this system was <90 min.

For an alternative approach to islet evaluation, we developed a metabolomic method to identify potential biomarkers of islet health for transplant. Using a miniaturized sample preparation method and HPLC-TOF-MS, we were able to identify 62 metabolites reliably in whole islet samples. To mimic damage that can occur during islet transplant, we induced oxidative stress in islets using hydrogen peroxide and measured their immediate metabolomic response as well as their response 1-4 h following stress removal. Increased concentrations of pentose phosphates, glucose-6-phosphate, and fructose bisphosphate in the immediate response corresponded to glycolysis blockage and possibly increased flux through the pentose phosphate pathway. Post-stress responses included increased levels of free fatty acids, phospholipids, long chain CoAs, and HMG-CoA as well blunted malonyl CoA concentrations, potentially relating to alterations in the glycerolipid/free fatty acid cycle and mevalonate pathway. These metabolites could comprise a metabolic signature of stressed cells for islet evaluation prior to transplantation.