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. 2019 Apr 26;12:108. doi: 10.3389/fnmol.2019.00108

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Copyright © 2019 Zhang, Hong, Cao, Yin, Shi and Ying.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The SIRT2 inhibitor AGK2 prevented the NAD⁺-induced increases in the glutathione levels, GSH/GSSG ratio, glutamylcysteine ligase (GCL) level and nuclear Nrf2 level of PC12 cells. (A) AGK2 prevented the NAD⁺-induced increase in the GSH levels (effect of NAD⁺ treatment, F_(1,36) = 15.91, p = 0.0003; effect of AGK2 treatment, F_(1,36) = 4.282, p = 0.0457; effect of AGK2 * NAD⁺ treatment, F_(1,36) = 6.875, p = 0.0127). (B) AGK2 prevented the NAD⁺-induced increase in the GSSG levels (effect of NAD⁺ treatment, F_(1,36) = 25.84, p < 0.0001; effect of AGK2 treatment, F_(1,36) = 2.402, p = 0.1299; effect of AGK2 * NAD⁺ treatment, F_(1,36) = 7.422, p = 0.0099). (C) AGK2 prevented the NAD⁺-induced increase in the GSH/GSSG ratio (effect of NAD⁺ treatment, F_(1,36) = 20.21, p < 0.0001; effect of AGK2 treatment, F_(1,36) = 8.883, p = 0.0051; effect of AGK2 * NAD⁺ treatment, F_(1,36) = 5.841, p = 0.0209). (D) AGK2 prevented the NAD⁺-induced increase in the total glutathione level (effect of NAD⁺ treatment, F_(1,36) = 4.292, p = 0.0455; effect of AGK2 treatment, F_(1,36) = 0.1002, p = 0.7534; effect of AGK2 * NAD⁺ treatment, F_(1,36) = 1.769, p = 0.1919). For (A–D), the assays were conducted after the cells were treated with 1 mM NAD⁺ and 5 μM AGK2 for 24 h. (E) AGK2 prevented the NAD⁺-induced increase in the GCLc mRNA level of the cells, assessed at 6 h after the NAD⁺ treatment (effect of NAD⁺ treatment, F_(1,44) = 15.46, p = 0.0003; effect of AGK2 treatment, F_(1,44) = 10.24, p = 0.0026; effect of AGK2 * NAD⁺ treatment, F_(1,44) = 15.59, p = 0.0003). (F) AGK2 prevented the NAD⁺-induced increase in the GCLm mRNA level of the cells, assessed at 6 h after the NAD⁺ treatment (effect of NAD⁺ treatment, F_(1,44) = 52.53, p < 0.0001; effect of AGK2 treatment, F_(1,44) = 9.449, p = 0.0036; effect of AGK2 * NAD⁺ treatment, F_(1,44) = 20.06, p < 0.0001). (G) Representative western blot bands of GCLc and GCLm. (H) AGK2 prevented the NAD⁺-induced increases in the protein levels of GCLc in PC12 cells, assessed at 12 h after the NAD⁺ treatment (effect of NAD⁺ treatment, F_(1,36) = 9.982, p = 0.0032; effect of AGK2 treatment, F_(1,36) = 5.051, p = 0.0245; effect of AGK2 * NAD⁺ treatment, F_(1,36) = 3.86, p = 0.0572). (I) AGK2 prevented the NAD⁺-induced increases in the protein levels of GCLm in PC12 cells, assessed at 12 h after the NAD⁺ treatment (effect of NAD⁺ treatment, F_(1,36) = 9.831, p = 0.0034; effect of AGK2 treatment, F_(1,36) = 2.646, p = 0.1125; effect of AGK2 * NAD⁺ treatment, F_(1,36) = 5.171, p = 0.029). (J) AGK2 prevented the NAD⁺-induced increase in the Nrf2 mRNA level of the cells, assessed at 6 h after the NAD⁺ treatment (effect of NAD⁺ treatment, F_(1,36) = 9.982, p = 0.0032; effect of AGK2 treatment, F_(1,36) = 5051, p = 0.0245; effect of AGK2 * NAD⁺ treatment, F_(1,36) = 3.86, p = 0.0572). (K) Representative western blot bands of nuclear Nrf2. (L) AGK2 prevented the NAD⁺-induced nuclear translocation of Nrf2, assessed at 1 h after the cells were treated with 1 mM NAD⁺ and 5 μM AGK2 (effect of NAD⁺ treatment, F_(1,32) = 3.753, p = 0.0616; effect of AGK2 treatment, F_(1,32) = 7.13, p = 0.0118; effect of AGK2 * NAD⁺ treatment, F_(1,32) = 10.28, p = 0.0030). The data were pooled from four independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.