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

Abstract

ObjectiveThe glucose stimulation of insulin secretion (GSIS) by pancreatic β-cells critically depends on increased production of metabolic coupling factors, including NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and ΔpH in energized mitochondria. Its spontaneous inactivation in C57BL/6J mice was previously shown to alter ATP production, Ca2+ influx, and GSIS, thereby leading to glucose intolerance. Here, we tested the role of NNT in the glucose regulation of mitochondrial NADPH and glutathione redox state and reinvestigated its role in GSIS coupling events in mouse pancreatic islets.MethodsIslets were isolated from female C57BL/6J mice (J-islets), which lack functional NNT, and genetically close C57BL/6N mice (N-islets). Wild-type mouse NNT was expressed in J-islets by adenoviral infection. Mitochondrial and cytosolic glutathione oxidation was measured with glutaredoxin 1-fused roGFP2 probes targeted or not to the mitochondrial matrix. NADPH and NADH redox state was measured biochemically. Insulin secretion and upstream coupling events were measured under dynamic or static conditions by standard procedures.ResultsNNT is largely responsible for the acute glucose-induced rise in islet NADPH/NADP+ ratio and decrease in mitochondrial glutathione oxidation, with a small impact on cytosolic glutathione. However, contrary to current views on NNT in β-cells, these effects resulted from a glucose-dependent reduction in NADPH consumption by NNT reverse mode of operation, rather than from a stimulation of its forward mode of operation. Accordingly, the lack of NNT in J-islets decreased their sensitivity to exogenous H2O2 at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca2+ influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca2+-induced exocytosis and its metabolic amplification.ConclusionThese results drastically modify current views on NNT operation and mitochondrial function in pancreatic β-cells.

Metabolic Responses to Reductive Stress

“…Interestingly, the increased ratio was caused by a suppression of the reverse operation of NNT enzyme rather than a stimulation of its forward operation since NADH/total NAD(H) was also elevated by the identical challenge. The rise in NAD(P)H levels correlated with enhancement of glucose-stimulated insulin secretion, glycolysis (as demonstrated by elevated levels of G-6-P and F-6-P), and mitochondrial respiration (as demonstrated by increases in glucose oxidation, oxygen consumption, ATP production, and the TCA cycle intermediate metabolites citrate, malate, and fumarate) (86). Collectively, NNT is therefore a key enzyme bridging cell metabolism and reductive stress.…”

“…In isolated pancreatic islets of C57BL/6N mice, glucose stimulation increased the NADPH/total NADP(H), which correlated with a reduction in mitochondrial GSH oxidation (86). Interestingly, the increased ratio was caused by a suppression of the reverse operation of NNT enzyme rather than a stimulation of its forward operation since NADH/total NAD(H) was also elevated by the identical challenge.…”

The pathogenetic role of β-cell mitochondria in type 2 diabetes

“…Recent studies in this substrain of C57BL/6J show that NNT is required for the acute glucose-induced rise in islet NADPH/NADP + ratio, while decreasing the mitochondrial glutathione oxidation (Santos et al 2017). This action depends on a reduction in NADPH consumption by NNT, which relies on glucose and appears to operate in reverse mode rather than from enhancing its forward mode of operation.…”

Nicotinamide nucleotide transhydrogenase is required for brain mitochondrial redox balance under hampered energy substrate metabolism and high‐fat diet

“…; Santos et al . ). Moreover, the absence of NNT activity in C57BL/6J mouse was shown to cause redox abnormalities in liver mitochondria, which are related to the poor ability to maintain NADP and glutathione in their reduced forms (Ronchi et al .…”

“…Mutations in the Nnt gene have been associated with adrenal insufficiency in humans and mice (Meimaridou et al 2012(Meimaridou et al , 2018Fujisawa et al 2015;Weinberg-Shukron et al 2015) and with dysfunctions in other organs (Metherell et al 2016;Roucher-Boulez et al 2016). A spontaneous Nnt mutation found in the widely used C57BL/6J mouse strain results in negligible NNT activity and is largely associated with impaired insulin secretion and glucose homeostasis (Toye et al 2005;Freeman et al 2006;Fergusson et al 2014;Santos et al 2017). Moreover, the absence of NNT activity in C57BL/6J mouse was shown to cause redox abnormalities in liver mitochondria, which are related to the poor ability to maintain NADP and glutathione in their reduced forms (Ronchi et al 2013).…”