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. 2018 May;15:115-124.
doi: 10.1016/j.redox.2017.09.007. Epub 2017 Sep 20.

Trehalose Protects Against Oxidative Stress by Regulating the Keap1-Nrf2 and Autophagy Pathways

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Free PMC article

Trehalose Protects Against Oxidative Stress by Regulating the Keap1-Nrf2 and Autophagy Pathways

Yuhei Mizunoe et al. Redox Biol. .
Free PMC article

Abstract

Dysfunction of autophagy, which regulates cellular homeostasis by degrading organelles and proteins, is associated with pathogenesis of various diseases such as cancer, neurodegeneration and metabolic disease. Trehalose, a naturally occurring nontoxic disaccharide found in plants, insects, microorganisms and invertebrates, but not in mammals, was reported to function as a mechanistic target of the rapamycin (mTOR)-independent inducer of autophagy. In addition, trehalose functions as an antioxidant though its underlying molecular mechanisms remain unclear. In this study, we showed that trehalose not only promoted autophagy, but also increased p62 protein expression, in an autophagy-independent manner. In addition, trehalose increased nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in a p62-dependent manner and enhance expression of its downstream antioxidant factors, heme oxygenase-1 (Ho-1) and nicotinamide adenine dinucleotide phosphate quinone dehydrogenase 1 (Nqo1). Moreover, treatment with trehalose significantly reduced amount of reactive oxygen species. Collectively, these results suggested that trehalose can function as a novel activator of the p62-Keap1/Nrf2 pathway, in addition to inducing autophagy. Therefore, trehalose may be useful to treat many chronic diseases involving oxidative stress and dysfunction of autophagy.

Keywords: Antioxidant; Autophagy; Keap1–Nrf2 system; Oxidative stress; Trehalose; p62.

Figures

Fig. 1
Fig. 1
Trehalose affects autophagy marker protein expression. (A) Hepa1-6 cells were treated with the indicated concentrations of trehalose for 6, 12 or 24 h. (B) Hepa1-6 cells were treated with 20 or 50 mM of various saccharides for 24 h. (C) Hepa1-6 cells were treated with the indicated concentrations of trehalose or sucrose for 24 h. (D) Atg5+/+ or Atg5-/- mouse embryonic fibroblasts (MEFs) were treated with the indicated concentrations of trehalose for 24 h. (E) p62+/+ or p62-/- MEFs were treated with the indicated concentrations of trehalose for 24 h. β-Actin was used as a loading control. Data are representative of two independent experiments.
Fig. 2
Fig. 2
Trehalose did not inhibit autophagic flux but accelerated p62 turnover. (A–C) Hepa1-6 cells were treated for 24 h with 50 or 100 mM trehalose, with or without chloroquine (CQ), while untreated cells were used as controls. Total cell lysates were analyzed by western blotting using anti-p62, LC3 and β-actin antibodies (A) and bands were quantified (B, C). β-Actin was used as a loading control. Representative images and quantitative data (n = 4) are shown. Values are means ± SD. Differences between values were analyzed by Student's t-test. Statistical significance shown as *p < 0.05, **p < 0.01. (D) Hepa1-6/RFP-GFP-LC3 cells were treated with 50 mM trehalose or 10 μM CQ for 8 h (upper panel). Hepa1-6 cells were treated with 50 mM trehalose or 10 μM CQ for 8 h and stained with LysoTracker Red for 30 min (lower panels). Images were acquired by confocal fluorescence laser microscopy. Scale bars are 20 µm and 50 µm, respectively. (E, F) Hepa1-6 shLuc (control)/shAtg5 cells were pretreated with 50 mM trehalose or 10 μM CQ for 24 h and then treated with 200 μM cycloheximide (CHX) for the indicated times. Total cell lysates were analyzed by western blotting using anti-p62 and LaminB1 antibodies and quantified. LaminB1 was used as a loading control. Representative images are shown (E). The quantitative data (n = 3) is shown as relative to the values at time 0 min for each cell type and under each experimental condition (F). Differences between values were analyzed by Student's t-test. Statistical significance shown as *p < 0.05, **p < 0.01.
Fig. 3
Fig. 3
Trehalose enhanced the effects of p62 expression and promoted Nrf2 nuclear translocation. (A) Hepa1-6 cells were treated with 50 mM sorbitol, sucrose or trehalose for 24 h, while untreated cells were used as a control. Total, nuclear and cytoplasmic protein extracts were prepared and analyzed by western blotting using anti-Nrf2, p62, p-p62 (S351), LaminB1 and α-tubulin antibodies. LaminB1 and α-tubulin were used as the loading controls for nuclear and cytoplasmic protein extracts, respectively. (B) Hepa1-6 cells were treated with 50 mM sorbitol, sucrose, or trehalose for 24 h and harvested, while untreated cells were used as a control. p62 mRNA expression was analyzed by real-time RT-PCR (n = 4). Data were normalized against Rps18 expression (n = 4). Values are means ± SD. Differences among values were analyzed by the Tukey-Kramer method with *p < 0.05, **p < 0.01. (C) p62+/+ or p62-/- mouse embryonic fibroblasts (MEFs) were treated with 50 mM trehalose for 24 h and harvested. Expression of p62 mRNA was analyzed by real-time RT-PCR (n = 4). Data were normalized against Rps18 (n = 4). Values are means ± SD. Differences between values were analyzed by Student's t-test. Statistical significance shown as *p < 0.05, **p < 0.01. (D) p62+/+ or p62-/- MEFs were treated with 50 mM sorbitol, sucrose or trehalose for 24 h. Untreated cells were used as a control. Nuclear and cytoplasmic protein extracts were prepared and analyzed by western blotting using anti-Nrf2, LaminB1 and α-tubulin antibodies. LaminB1 and α-tubulin were used as the loading controls for nuclear and cytoplasmic protein extracts, respectively. Data are representative of two independent experiments.
Fig. 4
Fig. 4
Trehalose induced Nrf2 nuclear translocation, predominantly through upregulation of p62 mRNA and protein expression. (A) p62+/+ mouse embryonic fibroblasts (MEFs) and p62-/- MEF stable cell lines were treated with 50 mM trehalose for 24 h and harvested. mRNA expression of p62 was analyzed by real-time RT-PCR (n = 4). Data were normalized against Rps18 (n = 4). Values are means ± SD. Differences among values were analyzed by the Tukey-Kramer method with *p < 0.05, **p < 0.01. (B–D) p62-/- MEFs were stably transfected with mock, wildtype p62 or p62 S351E mutant. The p62+/+ MEFs and p62-/- MEF stable cell lines were treated with 50 mM trehalose for 24 h and analyzed by western blotting using p62, p-p62 (S351E), LC3 and β-actin antibodies. Quantitative data for p62 are shown in (C). Quantitative data for p-p62 are shown as values excluding the background signals observed in mock p62-/- MEFs (D). β-Actin was used as a loading control. Representative images and quantitative data (n = 4) are shown. Values are means ± SD. Differences between values were analyzed by Student's t-test. Statistical significance shown as *p < 0.05, **p < 0.01. (E-F) p62-/- MEF stable cell lines were treated with 50 mM trehalose for 24 h. Nuclear and cytoplasmic protein extracts were prepared and analyzed by western blotting using anti-Nrf2, LaminB1 and α-tubulin antibodies. LaminB1 and α-tubulin were used as the loading controls for nuclear and cytoplasmic protein extracts, respectively. Representative images (E) and quantitative date (F) in Ratios between nuclear Nrf2 (nNrf2) and cytoplasmic Nrf2 (cNrf2) (n=3) are shown. Values are means ± SD. Differences between values were analyzed by Student's t-test. Statistical significance shown as *p < 0.05, **p < 0.01.
Fig. 5
Fig. 5
Trehalose exerted effects on the Keap1–Nrf2 pathway by altering p62 protein expression. (A-D) Hepa1-6 cells were treated with 50 mM trehalose for 24 h and harvested, while untreated cells served as a control. Expression of Ho-1 (A), Nqo1 (B), MnSOD (C) and Gpx4 (D) mRNA analyzed by real-time RT-PCR (n = 4). Data were normalized to Rps18 expression (n = 4). Values are means ± SD. Differences between values were analyzed by Student's t-test. Statistical significance shown as *p < 0.05, **p < 0.01.
Fig. 6
Fig. 6
Trehalose suppressed oxidative stress. (A, B) Hepa1-6 cells were pretreated with 50 mM trehalose or sucrose, or 10 mM N-acetylcysteine (NAC) for 24 h before incubation with 2 mM paraquat for 15 h. Intracellular ROS levels were analyzed with CM-H2DCFDA (n = 5). Values indicate means ± SD. Differences among values were analyzed by the Tukey-Kramer method with *p < 0.05, **p < 0.01.

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