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

Abstract

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 Ϸ30 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 Ϸ7 M. Likewise, the NAD(P)H increase of the mitochondrial domain is Ϸ60 M and is delayed with respect to the change in cytoplasmic NAD(P)H by Ϸ20 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.G lucose-induced insulin secretion is coupled to the metabolic state of the ␤ cell. After transport into the cell, glucose is phosphorylated and shunted into glycolysis, which increases metabolic flux. This altered metabolic state, which can be monitored by NAD(P)H autofluorescence increase, leads to an increase in the ATP͞ADP ratio that closes the plasma membrane-associated ATP-sensitive potassium (K ATP ) channel. Closure of this channel depolarizes the membrane, leading to the activation of voltage-sensitive calcium (Ca 2ϩ ) channels, Ca 2ϩ influx, and insulin secretion (1).Glucose usage in stimulated pancreatic ␤ cells is principally glycolytic, with the polyol pathway, glycogen synthesis, and pentose phosphate pathway accounting for Ͻ10% of total usage (2). Thus, the glucose metabolic signal is derived from glycolysis in the cytoplasm and pyruvate metabolism in the mitochondria. The absence of stimulated insulin secretion with nonmetabolizable glucose derivatives (1) and the abolition of glucosestimulated insulin secretion in a pancreatic ␤ cell line lacking mitochondrial DNA (3) suggest roles for both cytoplasmic and mitochondrial metabolism in normal secretion. Additionally, both glycolytic intermediates, such as glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (2), and mitochondrial substrates, such as leucine (4) or methyl pyruvate (5), stimulate insulin secretion, which further supports a role for metabolism in both compartments in glucose signaling.Attempts to resolve the glycolytic and mitochondrial contributions to glucose-stimulated insulin secretion have relied on nutrient secretagogues that couple at various points into glycolysis or Kreb's cycle or on pharmacological inhibition at various points along each pathway (1, 6). Although various nutrient supplements indicate that couplings of the pathway can lead to insulin secretion, they seldom mimic the effects of glucose. For example, pyruvate potentiates glucose-stimulated secretion but does not cause s...

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