Separation of the glucose-stimulated cytoplasmic mitochondrial NAD(P)H responses in pancreatic islet β cells

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be approximate to 30 mu M. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be approximate to 7 mu M. Likewise, the NAD(P)H increase of the mitochondrial domain is approximate to 60 mu M and is delayed with respect to the change in cytoplasmic NAD(P)H by approximate to 20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.