Abstract
We have previously demonstrated that islet depolarization with 70 mM KCl opens Cx36 hemichannels and allows diffusion of small metabolites and cofactors through the β-cell plasma membrane. We have investigated in this islet “permeabilized” model whether glycolytic and citric acid cycle intermediates stimulate insulin secretion and how it correlates with ATP production (islet content plus extracellular nucleotide accumulation). Glycolytic intermediates (10 mM) stimulated insulin secretion and ATP production similarly. However, they showed differential sensitivities to respiratory chain or enzyme inhibitors. Pyruvate showed a lower secretory capacity and less ATP production than phosphoenolpyruvate, implicating an important role for glycolytic generation of ATP. ATP production by glucose-6-phosphate was not sensitive to a pyruvate kinase inhibitor that effectively suppressed the phosphoenolpyruvate-induced secretory response and islet ATP rise. Strong suppression of both insulin secretion and ATP production induced by glucose-6-phosphate was caused by 10 μM antimycin A, implicating an important role for the glycerophosphate shuttle in transferring reducing equivalents to the mitochondria. Five citric acid cycle intermediates were investigated for their secretory and ATP production capacity (succinate, fumarate, malate, isocitrate and α-ketoglutarate at 5 mM, together with ADP and/or NADP+ to feed the NADPH re-oxidation cycles). The magnitude of the secretory response was very similar among the different mitochondrial metabolites but α-ketoglutarate showed a more sustained second phase of secretion. Gabaculine (1 mM, a GABA-transaminase inhibitor) suppressed the second phase of secretion and the ATP-production stimulated by α-ketoglutarate, supporting a role for the GABA shuttle in the control of glucose-induced insulin secretion. None of the other citric acid intermediates essayed showed any suppression of both insulin secretion or ATP-production by the presence of gabaculine. We propose that endogenous GABA metabolism in the “GABA-shunt” facilitates ATP production in the citric acid cycle for an optimal insulin secretion.
Highlights
According to the metabolic hypothesis, glucose has to be metabolized in the β-cells of pancreatic islets and increase the cytosolic ATP concentration and the ATP/ADP ratio in order to stimulate insulin secretion
We have previously proposed that αKG generated during glucose metabolism in the citric acid cycle might accumulate due to the limiting rate of αKG-dehydrogenase and diversion into the “GABA-shunt” where it would be transaminated to glutamate and semialdehyde succinic acid [10]
The secretory capacity of glycolytic intermediates participating in substrate-linked phosphorylation reactions was checked in rat islets permeabilized with 70 mM KCl and perifused at 5 mM glucose with the necessary additional cofactors for ATP synthesis
Summary
According to the metabolic hypothesis, glucose has to be metabolized in the β-cells of pancreatic islets and increase the cytosolic ATP concentration and the ATP/ADP ratio in order to stimulate insulin secretion. The resulting stimulation of insulin secretion was called the “triggering phase” [1] This phase may be reproduced at 5 mM glucose by β-cell membrane depolarization at a higher extracellular [KCl] than the physiological value, in the presence of diazoxide that keeps KATP-channels open, and non-responsive to ATP. It results in a transient increase of insulin secretion lasting for about 10 minutes before returning to the basal secretory level [1]. This second response was called the “amplifying phase” that is independent of KATP-channels because they remain open due to the continuous presence of diazoxide
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