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

Author(s)

Santos, Laila R. B. ; Muller, Carole ; de Souza, Arnaldo H. ; Takahashi, Hilton K. ; Spegel, Peter ; Sweet, Ian R. ; Chae, Heeyoung ; Mulder, Hindrik ; Jonas, Jean-Christophe

• Document type: Journal article
• Source: MOLECULAR METABOLISM; v. 6, n. 6, p. 535-547, JUN 2017.
• Web of Science Citations: 10

Abstract

Objective: The glucose stimulation of insulin secretion (GSIS) by pancreatic beta-cells critically depends on increased production of metaboliccoupling factors, including NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and Delta pH 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. Methods: Islets 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. Results: NNT 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 beta-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. Conclusion: These results drastically modify current views on NNT operation and mitochondrial function in pancreatic beta-cells. (C) 2017 The Authors. Published by Elsevier GmbH. (AU)

• FAPESP's process: 13/18232-5 - Role of NADPH oxidase on beta-cell function and survival after prolonged culture with different glucose concentrations
• Grantee: Arnaldo Henrique de Souza
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate