Elsevier

Molecular Metabolism

Volume 6, Issue 6, June 2017, Pages 535-547
Molecular Metabolism

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

https://doi.org/10.1016/j.molmet.2017.04.004Get rights and content
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open access

Abstract

Objective

The 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.

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 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.

Conclusion

These results drastically modify current views on NNT operation and mitochondrial function in pancreatic β-cells.

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Keywords

C57BL/6J mice
C57BL/6N mice
Glucose metabolism
GRX1-roGFP2
Insulin secretion
Mitochondrial shuttles
Pancreatic islet
Redox-sensitive GFP
Stimulus-secretion coupling

Abbreviations

AT2
aldrithiol
CMV
cytomegalovirus
Dz
diazoxide
DTT
dithiotreitol
FCCP
carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
GSIS
glucose stimulation of insulin secretion
GRX1
glutaredoxin 1
[Ca2+]i
intracellular Ca2+ concentration
IDH
isocitrate dehydrogenase
KRB
Krebs solution
ME
malic enzyme
WT
wild-type
NNT
nicotinamide nucleotide transhydrogenase
OCR
oxygen consumption rate

Cited by (0)

5

Laila R.B. Santos and Carole Muller contributed equally to this work.

6

Present address: Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany.

7

Present address: MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA.

8

Present address: Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Brazil.