SIRT2, ERK and Nrf2 Mediate NAD+ Treatment-Induced Increase in the Antioxidant Capacity of PC12 Cells Under Basal Conditions.

Jie Zhang,Weihai Ying,Haibo Shi,Yunyi Hong,Wei Cao,Shankai Yin

doi:10.3389/fnmol.2019.00108

Abstract

NAD+ (oxidized form of nicotinamide adenine dinucleotide) administration is highly beneficial in numerous models of diseases and aging. It is becoming increasingly important to determine if NAD+ treatment may directly increase the antioxidant capacity of cells under basal conditions. In the current study, we tested our hypothesis that NAD+ can directly enhance the antioxidant capacity of cells under basal conditions by using PC12 cells as a cellular model. We found that NAD+ treatment can increase the GSH/GSSG ratios in the cells under basal conditions. NAD+ can also increase both the mRNA and protein level of γ-glutamylcysteine ligase (γ-GCL)—a key enzyme for glutathione synthesis, which appears to be mediated by the NAD+-induced increase in Nrf2 activity. These NAD+-induced changes can be prevented by both SIRT2 siRNA and the SIRT2 inhibitor AGK2. The NAD+-induced changes can also be blocked by the ERK signaling inhibitor U0126. Moreover, the NAD+-induced ERK activation can be blocked by both SIRT2 siRNA and AGK2. Collectively, our study has provided the first evidence that NAD+ can enhance directly the antioxidant capacity of the cells under basal conditions, which is mediated by SIRT2, ERK, and Nrf2. These findings have suggested not only the great nutritional potential of NAD+, but also a novel mechanism underlying the protective effects of the NAD+ administration in the disease models: the NAD+ administration can enhance the resistance of the normal cells to oxidative insults by increasing the antioxidant capacity of the cells.

Highlights

NAD+ plays critical roles in energy metabolism, mitochondrial functions, calcium homeostasis and immune functions (Ying, 2005, 2008; Ma et al, 2012)
Our study has indicated that NAD+ can increase directly the GSH/GSSG ratio of PC12 cells by a γ-glutamylcysteine ligase (γ-GCL)dependent mechanism, which is mediated by SIRT2, ERK, and Nrf2
Our study determined the effects of NAD+ on the GSH/GSSG ratio and the levels of GSH, GSSG and total glutathione (GSH + GSSG) in the PC12 cells under basal conditions: treatment of the cells with 0.01, 0.1 and 1 mM NAD+ dose-dependently increased the GSH levels of the cells (Figure 1A), while only 1 mM NAD+ was capable of mildly increasing the GSSG levels (Figure 1B)

Summary

Introduction

NAD+ (oxidized form of nicotinamide adenine dinucleotide) plays critical roles in energy metabolism, mitochondrial functions, calcium homeostasis and immune functions (Ying, 2005, 2008; Ma et al, 2012). A number of studies have shown that NAD+ administration is highly beneficial in multiple models of major diseases (Ma et al, 2012). Multiple studies have shown that NAD+ treatment can decrease oxidative stress-induced death of several cell types including neurons, astrocytes and myocytes (Ying et al, 2003; Alano et al, 2004, 2010). Several studies have suggested that NAD+ treatment can indirectly decrease oxidative cell death by such mechanisms as enhancement of SIRT1 activity and prevention of glycolytic inhibition, mitochondrial permeability transition (MPT), mitochondrial depolarization and nuclear translocation of apoptosis-inducing factor (AIF; Alano et al, 2004, 2010; Araki et al, 2004; Hong et al, 2014)

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Results

Conclusion