Monitoring the [ATP]/[ADP] Ratio in Beta-Cells During Glucose Stimulated Insulin Secretion Using the Genetically Encoded Fluorescent Reporter Perceval

Abstract

Pancreatic beta-cells secrete insulin in response to elevated blood glucose levels. Glucose stimulated insulin secretion depends on glucose metabolism that produces ATP. The resulting increase in [ATP]/[ADP] ratio closes ATP-sensitive potassium (KATP) channels, which leads to membrane depolarization and opening of voltage-dependent Ca2+ channels. This causes an elevation of intracellular free Ca2+ and insulin exocytosis. Insulin is secreted in a pulsatile manner, which is thought to be regulated in part by oscillations in glucose metabolism. Such metabolic oscillations would also lead to oscillations in the [ATP]/[ADP] ratio and hence regulate KATP channel activity.Oscillations in [ATP]/[ADP] ratio have been demonstrated using biochemical and luciferase assays, but neither approach allows measurements of such oscillations in single cells. Perceval is a recently developed fluorescent protein biosensor for [ATP]/[ADP] ratio, and it permits direct measurement of [ATP]/[ADP] ratios inside living cells. We use Perceval in combination with quantitative confocal and two-photon excitation microscopy for direct measurement of the [ATP]/[ADP] ratio in beta-cells during glucose stimulated insulin secretion. For this purpose we have developed an adenoviral vector to express Perceval specifically in the beta-cells of intact mouse islets. Dynamic changes in [ATP]/[ADP] ratio can be correlated with glucose metabolism (by simultaneous imaging of Perceval fluorescence and NAD(P)H autofluorescence) and with intracellular free Ca2+ levels (by simultaneous imaging of Perceval fluorescence and the calcium sensor, FuraRed). This data allows us to test hypotheses regarding the role of localized subcellular signaling complexes and putative microdomains of glucose metabolism, [ATP]/[ADP] ratio, and Ca2+ dynamics in the regulation of glucose stimulated insulin secretion.