Abstract
Pancreatic beta-cell metabolism was followed during glucose and pyruvate stimulation of pancreatic islets using quantitative two-photon NAD(P)H imaging. The observed redox changes, spatially separated between the cytoplasm and mitochondria, were compared with whole islet insulin secretion. As expected, both NAD(P)H and insulin secretion showed sustained increases in response to glucose stimulation. In contrast, pyruvate caused a much lower NAD(P)H response and did not generate insulin secretion. Low pyruvate concentrations decreased cytoplasmic NAD(P)H without affecting mitochondrial NAD(P)H, whereas higher concentrations increased cytoplasmic and mitochondrial levels. However, the pyruvate-stimulated mitochondrial increase was transient and equilibrated to near-base-line levels. Inhibitors of the mitochondrial pyruvate-transporter and malate-aspartate shuttle were utilized to resolve the glucose- and pyruvate-stimulated NAD(P)H response mechanisms. These data showed that glucose-stimulated mitochondrial NAD(P)H and insulin secretion are independent of pyruvate transport but dependent on NAD(P)H shuttling. In contrast, the pyruvate-stimulated cytoplasmic NAD(P)H response was enhanced by both inhibitors. Surprisingly the malate-aspartate shuttle inhibitor enabled pyruvate-stimulated insulin secretion. These data support a model in which glycolysis plays a dominant role in glucose-stimulated insulin secretion. Based on these data, we propose a mechanism for glucose-stimulated insulin secretion that includes allosteric inhibition of tricarboxylic acid cycle enzymes and pH dependence of mitochondrial pyruvate transport.
Highlights
Pancreatic -cell metabolism was followed during glucose and pyruvate stimulation of pancreatic islets using quantitative two-photon NAD(P)H imaging
We propose a mechanism for glucose-stimulated insulin secretion that includes allosteric inhibition of tricarboxylic acid cycle enzymes and pH dependence of mitochondrial pyruvate transport
Because pyruvate is a mitochondrial fuel, the pyruvate dilemma has led to the suggestion that glycolysis and mitochondrial NADH shuttles play the primary role in glucose-stimulated insulin secretion (GSIS) (9)
Summary
Inhibitors of the mitochondrial pyruvate-transporter and malate-aspartate shuttle were utilized to resolve the glucose- and pyruvate-stimulated NAD(P)H response mechanisms. Ϳ90% of glucose-derived pyruvate enters the mitochondria for oxidation (11), it has been proposed that the tricarboxylic acid cycle of -cells does not actively metabolize pyruvate until Ca2ϩ-dependent dehydrogenases are “primed” by Ca2ϩ influx (10) In this model, glycolytic NADH induces a proton gradient across the mitochondrial membrane and stimulates mitochondrial Ca2ϩ influx. Two-photon NAD(P)H microscopy has proven successful for monitoring -cell redox state in pancreatic islets (1–3) This technique provides adequate spatial and temporal resolution to follow both cytoplasmic and mitochondrial NAD(P)H responses from individual -cells within intact islets during stimulated insulin secretion (1). The NAD(P)H response could be compared quantitatively with insulin secretion by islets in the presence and absence of these inhibitors
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