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

Two-photon excitation microscopy was used to image and quantify NAD(P)H aut ofluorescence from intact pancreatic islets under glucose stimulation. At m aximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estim ated to be approximate to 30 mu M. However, because glucose-stimulated insu lin secretion involves both glycolytic and Kreb's cycle metabolism islets w ere cultured on extracellular matrix that promotes cell spreading and allow s spatial resolution of the NAD(P)H signals from the cytoplasm and mitochon dria. The metabolic responses in these two compartments are shown to be dif ferentially stimulated by various nutrient applications. The glucose-stimul ated increase of NAD(P)H fluorescence within the cytoplasmic domain is esti mated to be approximate to 7 mu M. Likewise, the NAD(P)H increase of the mi tochondrial 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 mito chondrial change in glucose-stimulated NAD(P)H thus dominates the total sig nal but may depend on the smaller but more rapid cytoplasmic increase.