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

Santos, Laila R.B.; Muller, Carole; de Souza, Arnaldo H.; Takahashi, Hilton K.; Spégel, Peter LU ; Sweet, Ian R.; Chae, Heeyoung; Mulder, Hindrik LU and Jonas, Jean Christophe (2017) In Molecular Metabolism 6(6). p.535-547
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... (More)

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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author
organization
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type
Contribution to journal
publication status
published
subject
keywords
C57BL/6J mice, C57BL/6N mice, Glucose metabolism, GRX1-roGFP2, Insulin secretion, Mitochondrial shuttles, Pancreatic islet, Redox-sensitive GFP, Stimulus-secretion coupling
in
Molecular Metabolism
volume
6
issue
6
pages
13 pages
publisher
Elsevier GmbH
external identifiers
  • scopus:85018948800
  • wos:000405453000008
ISSN
2212-8778
DOI
10.1016/j.molmet.2017.04.004
language
English
LU publication?
yes
id
4b1f84f4-e8e1-4a7c-b281-81d540be229e
date added to LUP
2017-06-08 10:19:33
date last changed
2018-01-07 12:06:34
@article{4b1f84f4-e8e1-4a7c-b281-81d540be229e,
  abstract     = {<p>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, Ca<sup>2+</sup> 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<sup>+</sup> 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 H<sub>2</sub>O<sub>2</sub> at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca<sup>2+</sup> influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca<sup>2+</sup>-induced exocytosis and its metabolic amplification. Conclusion: These results drastically modify current views on NNT operation and mitochondrial function in pancreatic β-cells.</p>},
  author       = {Santos, Laila R.B. and Muller, Carole and de Souza, Arnaldo H. and Takahashi, Hilton K. and Spégel, Peter and Sweet, Ian R. and Chae, Heeyoung and Mulder, Hindrik and Jonas, Jean Christophe},
  issn         = {2212-8778},
  keyword      = {C57BL/6J mice,C57BL/6N mice,Glucose metabolism,GRX1-roGFP2,Insulin secretion,Mitochondrial shuttles,Pancreatic islet,Redox-sensitive GFP,Stimulus-secretion coupling},
  language     = {eng},
  number       = {6},
  pages        = {535--547},
  publisher    = {Elsevier GmbH},
  series       = {Molecular Metabolism},
  title        = {NNT reverse mode of operation mediates glucose control of mitochondrial NADPH and glutathione redox state in mouse pancreatic β-cells},
  url          = {http://dx.doi.org/10.1016/j.molmet.2017.04.004},
  volume       = {6},
  year         = {2017},
}