Acute nutrient regulation of the mitochondrial glutathione redox state in pancreatic β-cells

Abstract

The glucose stimulation of insulin secretion by pancreatic β-cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in β-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of EGSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in β-cells and reacted to low concentrations of exogenous H2O2 and menadione. Under control conditions, cytosolic/nuclear EGSH was close to -300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial EGSH was less negative and rapidly regulated by glucose and other nutrients, ranging from -280 mV in the absence of glucose to -299 mV in 30 mM glucose. These changes were largely independent from changes in intracellular Ca(2+) concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial EGSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, EGSH in pancreatic β-cells under control conditions.

Glucose-induced glutathione reduction in mitochondria is involved in the first phase of pancreatic β-cell insulin secretion

“…They also confirmed the observation in human islets [6]. But whether there is a relation between mitochondrial glutathione redox state and GSIS still remains unknown.…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…Increasing glucose produced a reductive shift in matrix thiol redox potential in adenoviral transfected rat islet clusters (504) or INS1-E cells (300). However, no glucose-dependent change in signal was detected from an untargeted, cytosolic, GRX1-roGFP2 probe (504). The implication is that the matrix NADP(H) and glutathione pools respond conventionally to changes in substrate supply, but that this is not transmitted into the cytosol within the time courses of these studies.…”

NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic β-cells

“…The generation and amplification of adenoviruses coding GRX1–roGFP2, mt-GRX1–roGFP2, or mt-SypHer under the control of the cytomegalovirus (CMV) promoter and of an adenovirus coding the red fluorescent protein DsRed under the control of the rat insulin promoter (Ad-RIP-DsRed) were described previously [11], [18], [19]. Adenoviruses coding WT mouse NNT together with mCherry (Ad-NNT) or mCherry alone (Ad-mCh) under the control of the CMV promoter were from Vector Biolabs (Philadelphia, PA).…”

“…Using the glutathione redox probe GRX1-roGFP2, we showed that the rise in NAD(P)H autofluorescence, which mainly occurs in mitochondria [10], correlates with a decrease in mitochondrial glutathione oxidation in rat and human β-cells, suggesting that mitochondrial NAD(P)H and glutathione redox state may play a role in GSIS [11]. Mitochondrial NADPH is typically produced by nicotinamide nucleotide transhydrogenase (NNT), isocitrate dehydrogenase (IDH) 2 and malic enzyme (ME) 3 [3], [12].…”