Extended oxygen consumption assay reveals size dependent differences of rat islets of Langerhans
Introduction: The mitochondrial viability of islet beta cells is critical for two key functions: first, the provision of energy for maintenance of cellular metabolism and homeostasis, and second, the induction of glucose stimulated insulin secretion. Recently the measurement of the oxygen consumption rate (OCR) in combination with the isolation factor, a marker of islet size, has been proposed as a new quality control parameter of islets destined for transplantation for the therapy of type 1 diabetes.
Methods: Islets isolated from female Wistar rats and visually sorted at the microscope in groups smaller and larger than 150 µm were investigated regarding their mitochondrial function. Specifically we determined their oxygen consumption in 3.3mM glucose using the Unisense MicroRespiration system, which allows for the injection of substances such a glucose, dinitrophenol and potassium cyanide, each stimulating or inhibiting different components of the respiratory chain and thereby differentially probing mitochondrial integrity. Glucose metabolism increases ATP synthesis in mitochondria and thus also their oxygen consumption. Dinitrophenol (DNP) disrupts instead the proton gradient across mitochondrial membranes, leading to maximal oxygen consumption. Potassium cyanide (KCN) inhibits mitochondrial complex 4, and can be used therefore to measure and subtract the non-mitochondrial respiration. Insulin secretion after glucose challenge was determined by ELISA.
Results: Our results show significant differences between islets smaller and larger than 150 µm. In comparison to larger islets smaller islets secrete a greater percentage of their total insulin. Smaller islets also display higher glucose induced oxygen consumption, higher maximal respiration after DNP and lower non-mitochondrial respiration after KCN in comparison to larger islets.
Conclusion: The data show differences in the mitochondrial function of smaller and larger islets. In larger islets the fraction of non-mitochondrial respiration is greater and less maximal respiration can be induced. This suggests a less efficient combustion of glucose to generate energy for beta cell specific functions such as insulin secretion. These data emphasize again the importance of measuring not only the islet particle number and the total islet equivalent, but also the islet size distribution prior to transplantation.