E 1, Hindrik Mulder 2, Jean-Christophe Jonas 1, * ABSTRACT Objective: The glucose stimulation of insulin secretion (GSIS) by pancreatic b-cells critically will depend on improved production of metabolic coupling components, which includes NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and DpH in energized mitochondria. Its spontaneous inactivation in C57BL/6J mice was previously shown to alter ATP production, Ca2influx, and GSIS, thereby top 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. Strategies: 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 to not the mitochondrial matrix. NADPH and NADH redox state was measured biochemically. Insulin secretion and upstream coupling events have been measured beneath dynamic or static conditions by common procedures. Final results: NNT is largely responsible for the acute glucose-induced rise in islet NADPH/NADPratio and decrease in mitochondrial glutathione oxidation, with a smaller impact on cytosolic glutathione. Having said that, contrary to present views on NNT in b-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.Cyclophilin A Protein Formulation Accordingly, the lack of NNT in J-islets decreased their sensitivity to exogenous H2O2 at non-stimulating glucose.Irisin Protein Purity & Documentation Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca2influx and upstream mitochondrial events, but it markedly lowered both phases of GSIS by altering Ca2induced exocytosis and its metabolic amplification.PMID:36717102 Conclusion: These results drastically modify current views on NNT operation and mitochondrial function in pancreatic b-cells.2017 The Authors. Published by Elsevier GmbH. This really is an open access report under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Keywords and phrases C57BL/6J mice; C57BL/6N mice; Glucose metabolism; GRX1-roGFP2; Insulin secretion; Mitochondrial shuttles; Pancreatic islet; Redox-sensitive GFP; Stimulus-secretion coupling 1. INTRODUCTION The glucose stimulation of insulin secretion (GSIS) by pancreatic bcells is determined by the enhanced flux of glucose metabolism by means of glycolysis and mitochondrial Krebs cycle, major to a rise inside the NADH/ NADratio, mitochondrial membrane hyperpolarization, matrix alkalization, and ATP synthesis. The ensuing rise inside the ATP/ADP ratio closes KATP channels, thereby depolarizing the plasma membrane, opening voltage-dependent Ca2channels and triggering Ca2influx, rise in intracellular Ca2concentration ([Ca2�]i) and insulin granule exocytosis. Also, glucose-derived coupling factors play a function in the metabolic amplification of Ca2induced secretion [1e4]. AmongUniversitcatholique de Louvain, Institute of Experimental and Clinical Research, Pole of Endocrinology, Diabetes and Nutrition, Brussels, B-1200, Belgium 2Lund University, Department of Clinical Sciences in Malm Unit of Molecular Metabolism, Malm 205 02, Sweden 3Lund University, Division of Chemistry, Centre for Evaluation and Synthes.
