Glucose Oxidation Depletes Cytosolic Citrate and Glutamate in the Amplifying Pathways of Insulin Secretion

In pancreatic β-cells, glucose metabolism generates signals that trigger and amplify insulin secretion. We generated β-cell specific FRET biosensors for the real-time measurement of cytosolic citrate and glutamate, two putative metabolic coupling factors with an undefined causal relationship to the signaling process. In islet β-cells, glucose elevation from 2.5 to 10 mM was accompanied by a sharp decrease in cytosolic citrate and glutamate. Steady-state oscillations in these intermediates induced by 10 mM glucose reveal that cytosolic citrate and glutamate levels decrease as calcium levels increase, precisely when the TCA cycle is most active. Subsequent membrane depolarization with 30 mM KCl resulted in a further decrease in citrate and glutamate, suggesting that mitochondria respond to work in the form of ATP hydrolysis by importing citrate and glutamate into the TCA cycle. Indeed, parallel measurements with Perceval-HR biosensors show that cytosolic ADP/ATP rises with calcium, and corresponds to a burst of mitochondrial NADH fluorescence. These data are inconsistent with existing models of the amplifying pathway in which mitochondrial glucose oxidation results in the export of TCA intermediates to the cytosol to generate NADPH and GSH/GSSG. The observed increase in cytosolic GSH/GSSG in response to glucose, shown with a FRET reporter targeted to β-cells, implies that GSH is generated from other sources such as the pentose phosphate pathway. Our results support the strong prediction that glucose oxidation in the mitochondria is inversely proportional to insulin secretion via the NADPH-generating citrate and glutamate cycles in the cytosol, since these pathways can never occur simultaneously.