- Email: [email protected]
Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity
CLS-08425; No of Pages 11 Cellular Signalling xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity Kyuhwa Seo 1, Sung Hwan Ki 1, Sang Mi Shin ⁎ College of Pharmacy, Chosun University, Gwangju 501-759, South Korea
a r t i c l e
i n f o
Article history: Received 28 January 2015 Accepted 3 March 2015 Available online xxxx Keywords: Glucose deprivation Sestrin2 Reactive oxygen species AMPK Mitochondria Hepatocytes
a b s t r a c t Sestrin2 (SESN2) regulates redox-homeostasis and apoptosis in response to various stresses. Although the antioxidant effects of SESN2 have been well established, the roles of SESN2 in mitochondrial function and metabolic stress have not yet been elucidated. In this study, we investigated the role of SESN2 in mitochondrial dysfunction under glucose deprivation and related signaling mechanisms. Glucose deprivation significantly upregulated SESN2 expression in hepatocyte-derived cells. Antioxidant treatments repressed SESN2 induction under glucose deprivation, this result suggested that reactive oxygen species (ROS) production was involved in SESN2 induction. Moreover, NF-E2-related factor-2 (Nrf2) phosphorylation was accompanied in induction of SESN2 by glucose deprivation. To elucidate the functional role of SESN2, we examined cells that stably overexpressed SESN2. Overexpression of SESN2 inhibited glucose deprivation-induced ROS production and cell death. In addition, under glucose deprivation, the changes in mitochondrial membrane potential, ADP/ATP ratio, and mitochondrial DNA content were significantly restored in SESN2-overexpressing cells. Moreover, siRNA knockdown of SESN2 failed to prevent mitochondrial permeability transition by glucose depletion. Mechanistic investigation showed that glucose deprivation significantly increased AMP-activated protein kinase (AMPK) activation. The recovery of mitochondrial function under glucose deprivation in SESN2-overexpressing cells was not seen in SESN2-overexpressing cells transfected with a dominant-negative AMPK; this result suggested that AMPK activation was responsible for SESN2-mediated mitochondrial protection against glucose deprivation. Treatment with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, an AMPK activator) also provided cytoprotective effects against glucose deprivation. Our findings provide evidence for the functional importance of SESN2–AMPK activation in the protection of mitochondria and cells against glucose deprivation-induced metabolic stress. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The sestrins (SESNs) are evolutionarily conserved stress-inducible genes that are involved in the complex regulation of cell survival in response to various stressful conditions such as genotoxic stress and oxidative stress [1,2]. In mammals, three kinds of SESNs (SESN1–3) have been characterized [2]. SESN1 and SESN2, initially known as PA26 and Hi95, respectively, were identified as p53 target genes and are known to regulate autophagy and cell viability [3,4]. SESN3 was identified as a
Abbreviations: AICAR, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK, AMP-activated protein kinase; AREs, antioxidant response elements; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; FoxOs, forkhead transcription factors; GSK3β, glycogen synthase kinase 3β; HO-1, heme oxygenase-1; mTOR, mammalian target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acetyl cysteine; Nrf2, NF-E2-related factor-2; PRX, peroxiredoxin; ROS, reactive oxygen species; SESN, sestrin; UCP, uncoupling protein ⁎ Corresponding author at: College of Pharmacy, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju 501-759, South Korea. Tel.: +82 62 230 6368; fax: +82 62 222 5414. E-mail address: [email protected] (S.M. Shin). 1 Both contributed equally to this work.
target gene for members of the forkhead transcription factor (FoxO) family and has been reported to affect the induction of SESN1 [5,6]. SESNs modulate redox-homeostasis via the regeneration of peroxiredoxins (PRXs), which controls hydrogen peroxide concentration under the diverse cellular processes [3,7]. Reactive oxygen species (ROS) inactivate PRXs via overoxidation of a catalytic cysteine residue. To sequester excessive ROS, PRXs must be restored via the sulfinyl reductase system. SESNs have been reported to play a critical role in the inhibition of intracellular ROS by rescuing of PRX from inactivation [7]. Recently, Shin et al. directly showed that SESN2 plays a critical role in cytoprotection under hydrogen peroxide-induced oxidative stress [8]. SESNs can also affect cell growth by controlling of mammalian target of rapamycin (mTOR) activity, but this function is not mediated by the modulation of redox activity [1,9]. mTOR is a master kinase complex that regulates protein synthesis, cell growth and metabolism. SESNs negatively regulate mTOR activity by AMP-activated protein kinase (AMPK) activation and TSC2 phosphorylation under genotoxic stress [1]. In addition, SESNs interact directly with AMPK in response to metabolic stress, thus helping to protect cells against stress-induced apoptosis [9]. In general, AMPK is activated by stimuli that increase the
http://dx.doi.org/10.1016/j.cellsig.2015.03.003 0898-6568/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
2
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
intracellular AMP/ATP ratio, and it inhibits energy-consuming processes and stimulates ATP-generating pathways in order to restore the energy balance [10,11]. Therefore, SESNs-mediated AMPK signaling has been proposed to play a crucial role in the regulation of cellular energy homeostasis and metabolism [2,12]. AMPK is activated in response to metabolic stress and plays a role in compensatory responses that protect cells from cell death by restoring mitochondrial function [11]. In our previous study, we reported that various AMPK activators such as resveratrol and 5-aminoimidazole-4carboxamide-1-β-D-ribofuranoside (AICAR) could protect cells from mitochondrial dysfunction-mediated apoptosis in hepatocytes [13,14]. Many reports have provided evidence that mitochondria are deeply involved in the regulation of metabolic stress-induced cell death [15]. Mitochondria are responsible for not only for ATP production, but also for the initiation of apoptosis [15]. The various cellular events in apoptosis such as loss of mitochondrial membrane potential and the subsequent release of cytochrome c are mediated in mitochondria [15]. Metabolic stress such as a decrease in the glucose supply results in the depletion of intracellular ATP level and sensitizes cells to apoptosis [16]. However, it is unclear whether SESNs, as upstream regulators of AMPK, could exert protective effects on mitochondria and cells. Metabolic stress changes electron transports in mitochondria and provokes the release of ROS [17,18]. Therefore, it has been reported to increase the expression of markers that indicate oxidative stress in cells [17,19]. Moreover, many enzymes responsible for redox signaling are induced as a major defense system against oxidative stress [20]. Heme oxygenase-1 (HO-1), an antioxidant gene whose expression is mediated by NF-E2-related factor-2 (Nrf2) and which is induced by glucose deprivation, has been shown to increase cell viability via the reduction of ROS production [21]. However, the possibility whether SESNs has protective effects against metabolic stress-induced apoptosis has not been reported. Based on the previous reports that SESN2 is an Nrf2-mediated antioxidant enzyme and activates AMPK, in this study, we investigated the role of SESN2–AMPK signaling in glucose depletion-induced apoptosis. We found that SESN2–AMPK signaling could exert a protective effect against glucose deprivation-induced cell death and that this effect is mediated by restoration of mitochondrial function. 2. Materials and methods 2.1. Materials Anti-SESN2 antibody was purchased from Proteintech (Chicago, IL). Anti-Nrf2 antibody, anti-PARP antibody, anti-Bcl − xL antibody, and rhodamine 123 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mn-TBAP was purchased from Cayman Chemical (Ann Arbor, MI). Cyclosporin A was purchased from Calbiochem (San Diego, CA). Caspase-3 antibody, p-ACC antibody, p-AMPK antibody, and AMPKα antibody were purchased from Cell Signaling (Danvers, MA). p-Nrf2 antibody was purchased from NOVUS Biologicals (Littleton, CO). Glucosefree Dulbecco's modified Eagle's medium (DMEM) was obtained from Life Technology (Gaithersburg, MD). Dimethylsulfoxide (DMSO), trolox, PEG-catalase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), anti-βactin antibody, rotenone, AICAR, N-acetyl cysteine (NAC) and other reagents were purchased from Sigma Chemicals (St. Louis, MO).
were maintained in media containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 50 units/mL penicillin and 50 μg/mL streptomycin, and cultured at 37 °C in a humidified atmosphere with 5% CO2. For experiments, cells were plated in plates for 2–3 days (i.e., 80% confluency) and serum starved overnight before treatments. 2.3. Establishment of a stable cell line expressing SESN2 Cells stably expressing SESN2 were established as previously described [22]. HepG2 cells were transfected with the plasmid pCMVTag3A (mock-transfected) or pCMV-SESN2 by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 1 day of transfection, colonies of geneticin-resistant cells were selected by treatment with geneticin (500 μg/mL) and then amplified in culture. SESN2 overexpression was established by immunoblotting. 2.4. Primary hepatocyte isolation Animal experiments were conducted under the guidelines of the Institutional Animal Use and Care Committee at Chosun University. Primary hepatocytes were isolated from male ICR mice (Samtako, Korea) according to standard procedures as follows [23]. Briefly, the mouse was injected with Zoletil (Virbac, France) for anesthesia and cannulated portal vein to perfuse the liver by HBSS containing 0.1% collagenase and calcium. After the liver was collected, it was minced gently with scissors. The minced tissue was suspended in DMEM, filtered through a cell strainer, and then centrifuged at 400 rpm for 5 min to separate parenchymal and nonparenchymal cells. Isolated hepatocytes were seeded on collagen-coated dishes and cultured in DMEM containing 75 units/mL penicillin and 75 μg/mL streptomycin with 10% FBS. Cell viability was determined via trypan blue staining; the viability of the isolated hepatocytes was usually 80–90%. 2.5. MTT assay Cells were seeded in a 48-well plate. For the assay, an MTT solution in PBS was added to each well, and the cells were incubated for 3 h. The resulting formazans were solubilized in DMSO, and their absorbance was measured at 570 nm using a microplate reader (Spectramax 190, Molecular Device, Sunnyvale, CA). Cell viability was calculated relative to the untreated control according to the following formula: viability (% control) = 100 × (absorbance of treated sample) / (absorbance of control). 2.6. Measurement of ROS generation The level of hydrogen peroxide production was determined by measuring the increase in dichlorofluorescein fluorescence after treatment with DCFH-DA, a cell-permeable nonfluorescent probe that is cleaved by intracellular esterases and oxidized primarily by hydrogen peroxide. Cells were stained with 10 μM DCFH-DA during the final hour of incubation. The cells were then harvested by trypsinization and washed twice with PBS. The intensity of the fluorescence in the cells was measured using a fluorescence microplate reader (Gemini XPS, Molecular Device, Sunnyvale, CA). ROS production was normalized to the protein concentration in each treated sample and calculated relative to the vehicletreated control.
2.2. Cell culture 2.7. Mitochondrial membrane permeability analysis The HepG2 and AML12 cell lines were purchased from the American Type Culture Collection (Manassas, VA). HepG2 cells were maintained in DMEM and Huh7 cells were maintained in RPMI. AML12 cells were cultured in 1:1 mixture of DMEM and Ham's F12 medium (Hyclone, Logan, UT) with 0.005 mg/mL insulin, 0.005 mg/mL transferrin, and 5 ng/mL selenium (Life Technologies, Gaithersburg, MD). All cells
Changes in mitochondrial membrane permeability were determined by staining with rhodamine 123, a membrane-permeable cationic fluorescent dye. Cells were treated with 0.05 μg/mL rhodamine 123 for last 1 h treatment. Sample collection and detection of fluorescence intensity were conducted as described in ROS production.
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
3
2.8. Immunoblot analysis
2.13. siRNA knockdown experiment
Cell lysates were prepared according to previously published procedures [13] and resolved using 7.5% or 12% gel electrophoresis. The proteins were then electophoretically transferred to nitrocellulose membranes. After the membranes were blocked, they were incubated with primary antibody at 4 °C overnight and then incubated with a secondary antibody. Protein bands of interest were identified using an ECL chemiluminescence system (GE Healthcare, Buckinghamshire, UK). Immunoblotting for β-actin confirmed equal loading of proteins.
Cells were transfected with either an siRNA directed against human SESN2 (Cat No. L-019134-02-0005, ON-TARGETplus SMARTpool; Dharmacon Inc., Lafayette, CO) or a non-targeting control siRNA (100 pmol/mL) using Lipofectamine 2000 (Invitrogen, San Diego, CA). After transfection for 24 h, cells were incubated in glucose (5.6 mM) or glucose-free DMEM. The knockdown of SESN2 was confirmed by immunoblot analysis. 2.14. Statistical analysis
2.9. RNA isolation and RT-PCR analysis RNA isolation was performed using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The RNA was then reverse transcribed using an oligo(dT)16 primer. PCR was conducted using a PCR premix (Bioneer, Daejeon, Korea) and a thermal cycler (Bio-Rad, Hercules, CA). GAPDH was used as a control for the amount of total mRNA. The following primer pairs were used for PCR: human SESN1 5′-CTTCTGGAGGCAGTTCAAGC-3′ (forward) and 5′-TGAATGGCAGCC TGTCTTCAC-3′ (reverse); human SESN2 5′-CTCACACCATTAAGCATG GAG-3′ (forward) and 5′-CAAGCTCGGAATTAATGTGCC-3′ (reverse); and human GAPDH 5′-GAAGATGGTGATGGGATTTC-3′ (forward) and 5′-GAAGGTGAAGGTCGGAGTC-3′ (reverse). 2.10. Plasmid transfection and luciferase assay A human SESN2 promoter-driven luciferase construct and dominant negative form of AMPK (D157A; DN-AMPK) were prepared as previously described [8,24]. Cells were plated in 12-well plates, incubated overnight, and then serum starved for 6 h. Transient transfection with the promoter-luciferase construct, pRL-TK plasmid (a plasmid that encodes for Renilla luciferase and is used to normalize transfection efficacy), and either DN-AMPK or pCDNA (control) was performed for 3 h using Lipofectamine (Invitrogen, San Diego, CA). The transfected cells were then incubated in minimum essential media containing 1% FBS for 14 h. Then, the luciferase activity in the lysates was determined using the dual-luciferase reporter assay system (Promega, Madison, WI) according to previously published procedures [8]. 2.11. Measurement of ADP/ATP ratio The ADP/ATP ratio was assessed using the EnzyLight ADP/ATP ratio assay kit (BioAssay Systems, Hayward, CA) according to the manufacturer's instructions. Briefly, the ATP reagent from the kit was added to cells grown in a 96-well plate. Then, luminescence due to ATP (RLU A) was measured using a luminometer (Promega, Madison, WI), and background luminescence (RLU B) was measured 10 min later. Next, ADP reagent was added to the cells and luminescence due to ADP (RLU C) was measured. Finally, the ADP/ATP was calculated by subtracting RLU B from RLU C and then dividing the result by RLU A. 2.12. Measurements of mitochondrial DNA Total DNA was extracted from cells according to the manufacturer's instructions (Nucleogen, Siheung, Korea). Levels of cytochrome c oxidase subunit II (mtCOX II) transcribed from mitochondrial DNA (mtDNA) were quantified by real-time PCR and normalized using nuclear-encoded receptor-interacting protein 140 (RIP140). The following primer pairs were used: human mtCOX II 5′-ACCTGCGACTCC TTGACGTTG-3′ (forward) and 5′-TAGGACGATGGGCATGAAACTG-3′ (reverse), and human RIP140 5′-GCTGGGCATAATGAAGAGGA-3′ (forward) and 5′-CAAAGAGGCCAGTAATGTGCTATC-3′ (reverse). Realtime PCR was performed using StepOne (Applied Biosystems, Foster City, CA) with a SYBR Green premix according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).
For each statistically significant effect of treatment, one-way analysis of variance (ANOVA) was used for comparisons between multiple group means. The data were expressed as means ± S.E. from at least three independent experiments. The criterion for statistical significance was set at p b 0.05 or p b 0.01. 3. Results 3.1. Glucose levels regulates SESN2 expression in hepatocytes SESN2 is an antioxidant enzyme that also regulates AMPK in response to various stresses [1,25]. Given that AMPK is a cellular energy sensor, we first examined whether SESN2 is regulated by glucose levels in hepatocytes. When we compared the level of SESN2 expression in primary hepatocytes isolated from mice that had been fasted for 18 h with that in hepatocytes isolated from nonfasted mice, we found that the level of SESN2 expression was higher in the hepatocytes from fasted mice (Fig. 1A, left). In hepatocyte-derived cell lines (AML12 and Huh7 cells), SESN2 induction in response to glucose deprivation was also observed (Fig. 1A, right). To further investigate the effect of glucose levels on SESN2 induction, HepG2 cells were incubated in media containing various concentrations (0–25 mM) of glucose, and then levels of SESN2 mRNA and protein expression were measured. When the cells were incubated in glucose-free media, levels of SESN2 mRNA and protein expression were increased (Fig. 1B and C). However, incubation of the cells in media containing high glucose (25 mM) did not significantly affect SESN2 expression when compared with normal glucose (5.6 mM). Moreover, the level of SESN1 mRNA expression remained unchanged (Fig. 1B). Incubation of cells with glucose-free media resulted in a marked increase in SESN2 mRNA expression starting from 6 h after the beginning of the experiment and peaking at 12 h, but the expression of SESN1 was not changed (Fig. 1D). A significant increase in SESN2 protein expression in cells incubated in glucose-free media was detected from 12 h after the start of the experiment and peaked at 18 h (Fig. 1E). To confirm that SESN2 was induced in response to glucose deprivation, cells were transiently transfected with a human SESN2 promoter-driven luciferase construct and then incubated in glucosefree medium. Glucose deprivation significantly increased the activity of the reporter construct; this result indicates that transcriptional induction of SESN2 was upregulated (Fig. 1F). Collectively, these results suggest that glucose deprivation increases the SESN2 induction. 3.2. Involvement of ROS production in glucose deprivation-induced SESN2 upregulation Previously, we found that SESN2 is induced by oxidative stress and knockdown of SESN2 promoted cell death mediated by hydrogen peroxide [8]. In addition, SESN2 has been shown to have cytoprotective activity against oxidative stress in various tissues [22,26,27]. Therefore, we investigated whether overproduction of ROS under glucose deprivation could lead to the induction of SESN2. First, we measured intracellular ROS accumulation by using DCFH-DA. When cells were incubated in glucose-free medium for 12 h, a significant increase in intracellular ROS levels was observed (Fig. 2A). To confirm that ROS induced SESN2
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
4
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
Fig. 1. Effect of glucose levels on Sestrin2 (SESN2) expression in hepatocytes-derived cells. (A) Immunoblot analysis. SESN2 protein level was determined by using lysates from primary hepatocytes from mice that had fasted for 18 h (left). AML12 and Huh7 cells were incubated in glucose-containing (5.6 mM) or glucose-free DMEM for 18 h (right). Lysates from these cells were then used to determine SESN2 protein level via immunoblotting. The blots shown are representative of data from at least 3 different replicates. (B) RT-PCR assays. HepG2 cells were incubated in the indicated concentrations of glucose for 6 h, and then the SESN1 or SESN2 transcripts were analyzed by RT-PCR. The results shown are representative of data from at least 3 different replicates. (C) Immunoblot analysis. HepG2 cells were incubated in the indicated concentrations of glucose for 12 h. Lysates from these cells were then used to determine SESN2 protein level via immunoblotting. The blots shown are representative of data from at least 3 different replicates. (D) RT-PCR assays. HepG2 cells were incubated in glucose-free DMEM for the indicated time period (0−12 h). SESN1 or SESN2 transcripts were then analyzed by RT-PCR. The results shown are representative of data from at least 3 different replicates; ⁎p b 0.05 or ⁎⁎p b 0.01 when compared to the control. (E) Immunoblot analysis. The lysates of cells incubated in glucose-free DMEM for 0−18 h were immunoblotted for SESN2 protein level. The blots shown are representative of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the control. (F) Luciferase activity. SESN2 luciferase activity was determined from the cell lysates incubated in glucose-free DMEM for 12 h. Data were expressed as the mean ± S.E. from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control.
expression, various antioxidants were added to the glucose-free medium. Treatment of the cells with NAC markedly inhibited the induction of SESN2 expression by glucose deprivation (Fig. 2B). In hepatocytes, mitochondria are the major sites for ROS production [28]. Hence, the
superoxide dismutase (SOD) mimetic Mn-TBAP was used to scavenge mitochondria-derived free radicals. The induction of SESN2 by glucose deprivation was suppressed by Mn-TBAP (Fig. 2C, left). Moreover, a low concentration of rotenone, a mitochondrial complex I inhibitor
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
5
Fig. 2. Involvement of ROS in glucose deprivation-induced SESN2 induction. (A) Measurement of ROS production. Cells were incubated in glucose (5.6 mM) or glucose-free DMEM for 12 h. ROS production was assessed by DCF fluorescent intensity. The data are expressed as mean ± S.E. from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control. (B) Immunoblot analysis. Cells were incubated with or without NAC (5 mM) in glucose (5.6 mM) or glucose-free DMEM for 12 h. Then, lysates from these cells were used to determine SESN2 protein level via immunoblotting. The blots shown are representative of data from at least 3 different replicates; ⁎p b 0.05 when compared to the glucose-incubated control; #p b 0.05 when compared to cells incubated in glucose-free DMEM without NAC. (C) Immunoblot analysis. Cells were incubated with or without Mn-TBAP (20 μM, left) or rotenone (10 nM, right) in glucose (5.6 mM) or glucose-free DMEM for 12 h. Then, lysates from these cells were used to determine SESN2 protein level via immunoblotting. The blots shown are representative of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; #p b 0.05 or ##p b 0.01 when compared to cells incubated in glucose-free DMEM without Mn-TBAP or rotenone.
which reduces ROS levels through inhibiting the mitochondrial respiratory chain [29], inhibited the induction of SESN2 by glucose deprivation (Fig. 2C, right); this result indicates that mitochondria-derived ROS are involved in the induction of SESN2 by glucose deprivation. Previously, we demonstrated that ROS-mediated SESN2 expression is mediated with Nrf2-antioxidant response element (ARE) activation [8]. In addition, Lee et al. showed that glucose deprivation increased nuclear translocation of Nrf2 and Nrf2–ARE-DNA binding in HepG2 cells [21]. To elucidate whether the induction of SESN2 by glucose deprivation was accompanied by Nrf2 activation, the phosphorylation of Nrf2 was observed. Incubation of cells in glucose-free medium increased the phosphorylation of Nrf2 (Fig. 3A). We also examined the functional role of ARE in SESN2 gene induction by using a ΔARESESN2 promoter. Specific disruption of the ARE in the promoter region
of the SESN2 gene significantly decreased the induction of SESN2 by glucose deprivation (Fig. 3B). These observations indicate that the Nrf2–ARE system contributes to the induction of SESN2 by glucose deprivation. Taken together, these results suggest that glucose deprivation produces ROS, which in turn upregulates SESN2 expression via Nrf2–ARE activation. 3.3. Involvement of mitochondrial damage in glucose deprivation-induced apoptosis In HepG2 cells, glucose deprivation significantly increases apoptotic cell death [21]. Since glucose deprivation leads to produce mitochondrial ROS and deplete intracellular ATP, glucose-depleted cells cannot maintain mitochondrial membrane permeability and the loss of mitochondrial
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
6
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
Fig. 3. Involvement of Nrf2 activation in glucose deprivation-induced SESN2 induction. (A) Immunoblot analysis. The lysates of cells incubated in glucose-free DMEM for 0−6 h were immunoblotted. The blots shown are representative of data from at least 3 different replicates; ⁎p b 0.05 when compared to the control. (B) Luciferase activity. Cells had been transfected with pGL4-phSESN2 or pGL3-phSESN2-ΔARE and incubated in glucose (5.6 mM) or glucose-free DMEM for 12 h. Luciferase activity was determined from the cell lysates. Data were expressed as the mean ± S.E. from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control in pGL4-phSESN2 transfected cells; ##p b 0.01 when compared to cells incubated in glucose-free DMEM after pGL4-phSESN2 transfection.
membrane permeability activates the apoptotic signal cascade [16,30]. Therefore, in an attempt to correlate mitochondrial damage with glucose deprivation-induced apoptosis, mitochondrial membrane permeability was measured using rhodamine 123 staining [31]. When cells were incubated in glucose-free medium, the intensity of rhodamine 123 fluorescence significantly decreased; this result indicates that glucose deprivation induces mitochondrial membrane damage (Fig. 4A). In accordance with previous reports that cyclosporin A prevents mitochondrial damage by inhibiting mitochondrial membrane transition pore formation [14,32,33], the addition of cyclosporin A was found to restore the loss of membrane permeability induced by glucose deprivation (Fig. 4A). In addition, cyclosporin A significantly prevented glucose deprivation-induced
Fig. 4. Involvement of mitochondrial dysfunction in glucose deprivation-induced apoptosis. (A) Measurement of mitochondrial membrane permeability changes. Cells were incubated in the indicated DMEM with or without cyclosporin A (10 μg/mL) for 18 h. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; #p b 0.05 when compared to cells incubated in glucose-free DMEM without cyclosporin A. (B) Cell viability assay. Cells were incubated in indicated DMEM without or with cyclosporin A (10 μg/mL) for 24 h. The effect of cyclosporin A on cell viability was then assessed using MTT assay. Data are expressed as the means ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; ##p b 0.01 when compared to cells incubated in glucose-free DMEM without cyclosporin A. (C) Cell viability assay. Cells were incubated in indicated DMEM with or without trolox (100 μM), PEG-catalase (catalase, 1000 U/mL), or Mn-TBAP (20 μM) for 24 h. The effect of these antioxidants on cell viability was then assessed using the MTT assay. The data are expressed as the means ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; ##p b 0.01 when comparing treatment groups under glucose-free DMEM incubation.
cell death (Fig. 4B). Moreover, trolox (vitamin E analog and hydroxyl radical scavenger), catalase (a hydrogen peroxide scavenger), and Mn-TBAP also prevented glucose deprivation-induced cell death from glucose
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
7
deprivation (Fig. 4C); this result suggests that intracellular ROS, including mitochondrial ROS, play an important role in apoptosis. Taken together, these results provide evidence that glucose deprivation-induced ROS cause mitochondrial damage, which subsequently lead to cell death. 3.4. Prevention of glucose deprivation-induced cytotoxicity by SESN2 overexpression To determine whether SESN2 has a cytoprotective effects, we prepared cell lines that stably overexpressed SESN2; SESN2 overexpression was confirmed by western blotting (Fig. 5A, upper). In comparison to the cells which had been stably transfected with pCMV-Tag3A (mocktransfected cells), SESN2 overexpression repressed glucose deprivationinduced cell death (Fig. 5A, lower). The protective effects of SESN2 were confirmed by observing proteins associated with apoptosis. PARP cleavage, caspase-3 activation, and a decrease in Bcl − xL expression, all of which were induced by glucose deprivation, did not occur in SESN2overexpressing cells (Fig. 5B). Moreover, glucose deprivation-induced ROS production was reduced in SESN2-overexpressing cells (Fig. 5C). Overall, these results indicate that SESN2 protects cells against glucose deprivation-induced apoptosis by reducing ROS production. 3.5. Inhibition of glucose deprivation-induced mitochondrial damage by SESN2 overexpression Next, we determined whether SESN2 could affect mitochondrial function under glucose deprivation. Since mitochondria play a central role in intracellular ATP production, the ADP/ATP ratio was used as a measure of mitochondrial function. ADP/ATP ratio was increased in mock-transfected cells by glucose deprivation. In contrast, the ADP/ ATP ratio of SESN2-overexpressing cells was decreased when compared with mock-transfected cells (Fig. 6A). In addition, glucose deprivation in mock-transfected cells significantly decreased levels of mtDNA, which represents numbers of mitochondria, but mtDNA levels were restored in SESN2-overexpressing cells (Fig. 6B). Moreover, rhodamine 123 fluorescence intensity under glucose deprivation was significantly higher in SESN2-overexpressing cells than in mock-transfected cells; this result suggests that SESN2 helps to preserve the mitochondrial membrane potential (Fig. 6C). Similarly, transfection with an siRNA directed against human SESN2 potentiated the glucose deprivation-induced loss of mitochondrial permeability (Fig. 6D, lower); SESN2 knockdown was confirmed by immunoblot analysis (Fig. 6D, upper). These results indicate that SESN2 plays a critical role in the maintenance of mitochondrial function under glucose deprivation-induced oxidative stress. 3.6. The role of AMPK activation in the recovery of mitochondrial function Previous studies have shown that SESN2 regulates AMPK activity, which activates an adaptive response during various stresses [1,9,25]. In view of the fact that AMPK is a sensor of energy status, we examined the role of the SESN2–AMPK axis in protecting mitochondria during glucose deprivation. Glucose deprivation increased phosphorylation of ACC, which represents cellular AMPK activity (Fig. 7A). The increase of ACC phosphorylation by glucose deprivation in SESN2-overexpressing cells was similar to mock-transfected cells (Fig. 7B). Next, we investigated the role of AMPK activation in SESN2 mediated-protective effects in mitochondria. The recovery of fluorescence intensity elicited by SESN2 overexpression was significantly reversed by DN-AMPK overexpression (Fig. 7C, lower). Overexpression of the DN-AMPK was verified by observation of AICAR-induced ACC phosphorylation (Fig. 7C, upper). To assess whether AMPK activation could protect cells against glucose deprivation, an MTT assay was conducted on cells treated with AICAR, an AMPK activator. AICAR treatment resulted in a notable increase in cell viability in glucose-deprived cells (Fig. 7D). These results strongly support the hypothesis that the protection provided by SESN2 against
Fig. 5. Inhibition of glucose deprivation-induced apoptosis by SESN2 overexpression. (A) Cell viability assay. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 24 h. Cell death was determined by the MTT assay. Data represent the mean ± S.E. of at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucoseincubated mock-transfected cells; ##p b 0.01 when comparing mock-transfected cells and SESN2-overexpressing cells in glucose free-DMEM. (B) Immunoblot analysis. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 24 h. Then, lysates from these cells were used to determine levels of apoptotic proteins via immunoblotting. The blots shown are representative of data from at least 3 different replicates. (C) Measurement of ROS generation. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 12 h. ROS levels were then assessed via DCF fluorescent intensity. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated mock-transfected cells; #p b 0.05 when comparing mock-transfected cells in glucose-free DMEM and SESN2-overexpressing cells in glucose free-DMEM.
glucose deprivation-induced mitochondria damage and cell death might be linked to AMPK activation. 4. Discussion In the present study, we show that increased ROS production caused by glucose deprivation induces SESN2, which protects mitochondria
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
8
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
Fig. 6. Inhibition of mitochondrial dysfunction by SESN2 overexpression. (A) ADP/ATP ratio assay. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 15 h. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎p b 0.05 when compared to glucose-incubated mock-transfected cells; ##p b 0.01 when comparing mocktransfected cells and SESN2-overexpressing cells in glucose free-DMEM. (B) mtDNA contents. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 15 h. The samples were then subjected to real-time PCR analysis with primers for the mtDNA region COXII. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated mock-transfected cells; ##p b 0.01 when comparing mock-transfected cells and SESN2-overexpressing cells in glucose free-DMEM. (C) Measurement of mitochondrial membrane permeability changes. Cells were incubated in DMEM containing glucose (5.6 mM) or glucose-free DMEM for 18 h. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated mock-transfected cells; ##p b 0.01 when comparing mock-transfected cells and SESN2-overexpressing cells in glucose free-DMEM. (D) Measurement of mitochondrial membrane permeability changes. Cells were transfected with control siRNA (siRNA CON) or SESN2 siRNA (siRNA SESN2). Then, cells were incubated in DMEM containing glucose (5.6 mM) or glucose-free DMEM for 12 h; ⁎p b 0.05 when compared to glucose-incubated control siRNA-transfected cells; #p b 0.05 when comparing control siRNA-transfected cells and SESN2 siRNA-transfected cells in glucose free-DMEM.
and cells from metabolic stress. In addition, our results suggest that SESN2–AMPK activation under energy depletion plays a crucial role in the adaptive survival response. The beneficial roles of SESNs are well documented. p53-Mediated SESN1 and SESN2 induction inhibits mTOR signaling via AMPK activation and TSC phosphorylation, and thus leads to metabolic arrest [1,9, 34]. SESNs have been demonstrated to induce autophagy, which leads to protect neuron and renal tubules against cytoxicity [26,35]. In addition, SESN2 attenuates high glucose-induced dysfunction of endothelial nitric oxide synthase and synthesis of fibronectin in glomerular mesangial cells [36]. Loss of epithelial barrier integrity and muscle degeneration is also prevented by SESN2 induction [37,38]. Moreover, SESN2 is involved in lipid lowering effects by downregulation of liver X receptor-α-dependent lipogenic gene expression [23]. Our study shows that SESN2 has an additional, crucial role in mitochondrial protection under hyponutrition. Collectively, SESNs have been demonstrated to show beneficial effects against oxidative injury, lipid accumulation, and tissue degeneration.
Three isoforms of SESNs (SESN1–3) have been reported in mammals. Although all three isoforms have been reported to decrease intracellular ROS, the induction of each SESN is regulated by different mechanisms. In general, SESN1 and SESN2 are induced by various stresses, which are mediated with p53 activation. SESN3 expression is regulated by Akt and FoxO-mediated signaling, but not induced by treatment with 2-deoxyglucose (an inhibitor of glycolysis) [9]. Here, we show that expression of SESN2 but not SESN1 is induced by glucose deprivation in HepG2 cells (Fig. 1). Bae et al., also observed that fasting and refeeding states regulate SESN2 level in mouse liver [39]. Moreover, we also observe that ROS play a critical role in SESN2 induction under glucose deprivation (Fig. 2). In accordance with present study, previous study reported that tert-butyl hydroquinone caused SESN2 induction but not SESN1 or SESN3 in HepG2 cells [8]. Collectively, these results indicate that among three isoforms of SESNs, SESN2 is sensitively induced under oxidative and metabolic stress in hepatocytes-derived cells. In a report from Ahmad et al., glucose depletion increases representative markers of oxidative stress [17]. Indeed, cellular antioxidants
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
9
Fig. 7. Involvement of AMP-activated protein kinase (AMPK) activation in SESN2-mediated mitochondrial protection. (A) Immunoblot analysis. The lysates of cells that had been incubated in glucose-free DMEM for 0–18 h were immunoblotted. The blots shown are representative of data from at least 3 different replicates; ⁎p b 0.05 or ⁎⁎p b 0.01 when compared to the control. (B) Immunoblot analysis. Cells were incubated in DMEM containing glucose (5.6 mM) or glucose-free DMEM for 12 h. Protein levels in lysates from these cells were then determined via immunoblotting. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated each control cells. (C) Measurement of mitochondrial membrane permeability changes. Mock-transfected or SESN2-overexpressing cells were transfected with a construct expressing a dominant-negative form of AMPK (DN-AMPK) or pCDNA (i.e., empty plasmid). Then, cells were incubated in DMEM containing glucose (5.6 mM) or glucose-free DMEM for 12 h. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated pCDNA and mock-transfected cells; ##p b 0.01 when comparing pCDNA and mock-transfected cells and pCDNA transfected SESN2 overexpressing cells in glucose free-DMEM. (D) Cell viability assay. Cells were incubated with or without AICAR (2 mM) in indicated DMEM for 24 h. The effect of AICAR on cell viability was then assessed using the MTT assay. The data were expressed as means ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; ##p b 0.01 when compared to cells incubated in glucose-free DMEM without AICAR.
including HO-1 and catalase are induced by exposure to glucose-free medium [21,40]. This induction is accompanied by an increase in ROS production [21,40]. It is well established that ROS induce a large number of antioxidant genes by activation of ARE and transcriptional modulation of genes [8,21,41]. Nrf2 is the transcription factor that activates ARE-mediated antioxidant gene expression [41]. Previous study by Lee
et al. reported that nuclear translocation of Nrf2 and Nrf2–ARE-DNA binding were increased by glucose deprivation, which contributed to HO-1 induction [21]. Since translocation or stabilization of Nrf2 is regulated by phosphoylation [42], we observe Nrf2 phosphorylation under glucose deprivation (Fig. 3A). The phosphorylation of Nrf2 Ser40 has been reported to result in the escape or release of Nrf2 from Kelch-like
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
10
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
mitochondrial dysfunction and cell death (Fig. 8). These findings provide novel insights into the mechanisms underlying metabolic stress-mediated cellular signaling and into the importance of SESN2 in the cellular adaptive response. Acknowledgment This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No. R13-2008-010-00000-0). References
Fig. 8. Schematic diagram illustrating that SESN2 inhibits mitochondrial dysfunction and apoptosis against glucose deprivation-induced stress by AMPK activation.
ECH-associated protein 1, an inhibitory regulator of Nrf2 [43]. Therefore, increase of Nrf2 Ser40 phosphorylation by glucose deprivation represents Nrf2 activation. In addition, we show that SESN2 induction is mediated with Nrf2–ARE activation as evidenced by SESN2 luciferase activity containing ARE or not (Fig. 3B). Another study by Ben-Sahra et al. demonstrated that ATP depletion upregulated SESN2 level in an Akt-dependent but p53-independent manner [9]. In view of the fact that hypoxic conditions induce SESN2 [3], glucose deprivation-induced hypoxia-inducible factor-1α might contribute to upregulate SESN2 [44]. Further detailed study is needed to understand the mechanisms underlying the transcriptional regulation of SESN2 in response to metabolic stress. The evidence indicating a direct link between SESN2 and mitochondria remains questionable. In adipose tissues, the antioxidant activity of SESN2 contributes to inhibit mitochondrial uncoupling protein (UCP) expression and plays a critical role in fat metabolism [45]. Here, we show that the protective effects on mitochondria by SESN2 require AMPK activation. Therefore, downstream targets of AMPK probably play a crucial role in SESN2-mediated mitochondrial protection. The primary regulator of AMPK activity is the cellular AMP/ATP ratio [10]. In response to an increase in the AMP/ATP ratio, AMPK represses ATPconsuming processes and increases ATP synthesis [11]. Since mitochondria are the sites of ATP production, mitochondria may be target of AMPK to preserve cellular energy content [11]. It is well accepted that AMPK suppresses ROS accumulation in mitochondria by increasing the levels of mRNA expression for peroxisome proliferators-activated response-coactivator-1, UCP, and MnSOD [46]. AMPK activation is also able to regulate mitochondrial permeability by inhibiting glycogen synthase kinase 3β (GSK3β). GSK3β activated by oxidative stress can translocate into the mitochondria, where it phosphorylate components of the permeability transition pore and induces the collapse of membrane potential [47,48]. Therefore, the inhibitory phosphorylation of GSK3β by AMPK contributes to mitochondrial protection. Moreover, because both SESN2 and AMPK negatively regulate mTOR activity, ATP-consuming processes by mTOR can be reduced to help maintain ATP levels under metabolic stress. 5. Conclusion All together, we have shown that SESN2–AMPK activation inhibits glucose deprivation-induced ROS production, thereby preventing
[1] A.V. Budanov, M. Karin, Cell 134 (2008) 451–460. [2] A.V. Budanov, J.H. Lee, M. Karin, EMBO Mol. Med. 2 (2010) 388–400. [3] A.V. Budanov, T. Shoshani, A. Faerman, E. Zelin, I. Kamer, H. Kalinski, S. Gorodin, A. Fishman, A. Chajut, P. Einat, R. Skaliter, A.V. Gudkov, P.M. Chumakov, E. Feinstein, Oncogene 21 (2002) 6017–6031. [4] M.C. Maiuri, S.A. Malik, E. Morselli, O. Kepp, A. Criollo, P.-L. Mouchel, R. Carnuccio, G. Kroemer, Cell Cycle 8 (2009) 1571–1576. [5] V. Nogueira, Y. Park, C.-C. Chen, P.-Z. Xu, M.-L. Chen, I. Tonic, T. Unterman, N. Hay, Cancer Cell 14 (2008) 458–470. [6] H. Tran, A. Brunet, J.M. Grenier, S.R. Datta, A.J. Fornace, P.S. DiStefano, L.W. Chiang, M.E. Greenberg, Science 296 (2002) 530–534. [7] A.V. Budanov, A.A. Sablina, E. Feinstein, E.V. Koonin, P.M. Chumakov, Science 304 (2004) 596–600. [8] B.Y. Shin, S.H. Jin, I.J. Cho, S.H. Ki, Free Radic. Biol. Med. 53 (2012) 834–841. [9] I. Ben-Sahra, B. Dirat, K. Laurent, A. Puissant, P. Auberger, A. Budanov, J.F. Tanti, F. Bost, Cell Death Differ. 20 (2013) 611–619. [10] D.G. Hardie, Nat. Rev. Mol. Cell Biol. 8 (2007) 774–785. [11] G.R. Steinberg, B.E. Kemp, Physiol. Rev. 89 (2009) 1025–1078. [12] J.H. Lee, A.V. Budanov, S. Talukdar, E.J. Park, H.L. Park, H.-W. Park, G. Bandyopadhyay, N. Li, M. Aghajan, I. Jang, A.M. Wolfe, G.A. Perkins, M.H. Ellisman, E. Bier, M. Scadeng, M. Foretz, B. Viollet, J. Olefsky, M. Karin, Cell Metab. 16 (2012) 311–321. [13] S.M. Shin, I.J. Cho, S.G. Kim, Mol. Pharmacol. 76 (2009) 884–895. [14] S.M. Shin, S.G. Kim, Mol. Pharmacol. 75 (2009) 242–253. [15] D.R. Green, J.C. Reed, Science 281 (1998) 1309–1312. [16] K.H. Moley, M.M. Mueckler, Apoptosis 5 (2000) 99–105. [17] I.M. Ahmad, N. Aykin-Burns, J.E. Sim, S.A. Walsh, R. Higashikubo, G.R. Buettner, S. Venkataraman, M.A. Mackey, S.W. Flanagan, L.W. Oberley, D.R. Spitz, J. Biol. Chem. 280 (2005) 4254–4263. [18] E. Cadenas, K.J.A. Davies, Free Radic. Biol. Med. 29 (2000) 222–230. [19] J.D. Browning, J.D. Horton, J. Clin. Invest. 114 (2004) 147–152. [20] J.L. Evans, I.D. Goldfine, B.A. Maddux, G.M. Grodsky, Endocr. Rev. 23 (2002) 599–622. [21] H.G. Lee, M.H. Li, E.J. Joung, H.K. Na, Y.N. Cha, Y.J. Surh, Antioxid. Redox Signal. 13 (2010) 1639–1648. [22] K. Seo, S. Seo, J.Y. Han, S.H. Ki, S.M. Shin, Toxicol. Appl. Pharmacol. 280 (2014) 314–322. [23] S.H. Jin, J.H. Yang, B.Y. Shin, K. Seo, S.M. Shin, I.J. Cho, S.H. Ki, Toxicol. Appl. Pharmacol. 271 (2013) 95–105. [24] S.H. Hwahng, S.H. Ki, E.J. Bae, H.E. Kim, S.G. Kim, Hepatology 49 (2009) 1913–1925. [25] T. Sanli, K. Linher-Melville, T. Tsakiridis, G. Singh, PLoS One 7 (2012) e32035. [26] Y.-S. Chen, S.-D. Chen, C.-L. Wu, S.-S. Huang, D.-I. Yang, Exp. Neurol. 253 (2014) 63–71. [27] Y. Yang, S. Cuevas, S. Yang, V.A. Villar, C. Escano, L. Asico, P. Yu, X. Jiang, E.J. Weinman, I. Armando, P.A. Jose, Hypertension 64 (2014) 825–832. [28] J.J. Lemasters, A.L. Nieminen, Biosci. Rep. 17 (1997) 281–291. [29] A.V. Gyulkhandanyan, P.S. Pennefather, J. Neurochem. 90 (2004) 405–421. [30] C.J. De Saedeleer, P.E. Porporato, T. Copetti, J. Perez-Escuredo, V.L. Payen, L. Brisson, O. Feron, P. Sonveaux, Oncogene 33 (2014) 4060–4068. [31] Y.N. Kwon, S.M. Shin, I.J. Cho, S.G. Kim, Drug Metab. Dispos. 37 (2009) 1187–1197. [32] V. Petronilli, D. Penzo, L. Scorrano, P. Bernardi, F. Di Lisa, J. Biol. Chem. 276 (2001) 12030–12034. [33] L. Scorrano, D. Penzo, V. Petronilli, F. Pagano, P. Bernardi, J. Biol. Chem. 276 (2001) 12035–12040. [34] J.H. Lee, A.V. Budanov, M. Karin, Cell Metab. 18 (2013) 792–801. [35] M. Ishihara, M. Urushido, K. Hamada, T. Matsumoto, Y. Shimamura, K. Ogata, K. Inoue, Y. Taniguchi, T. Horino, M. Fujieda, S. Fujimoto, Y. Terada, Am. J. Physiol. Lung Cell. Mol. Physiol. 305 (2013) F495–F509. [36] A.A. Eid, D.-Y. Lee, L.J. Roman, K. Khazim, Y. Gorin, Mol. Cell. Biol. 33 (2013) 3439–3460. [37] J.H. Lee, A.V. Budanov, E.J. Park, R. Birse, T.E. Kim, G.A. Perkins, K. Ocorr, M.H. Ellisman, R. Bodmer, E. Bier, M. Karin, Science 327 (2010) 1223–1228. [38] N. Olson, M. Hristova, N.H. Heintz, K.M. Lounsbury, A. van der Vliet, Am. J. Physiol. Lung Cell. Mol. Physiol. 301 (2011) L993–L1002. [39] S.H. Bae, S.H. Sung, S.Y. Oh, J.M. Lim, S.K. Lee, Y.N. Park, H.E. Lee, D. Kang, S.G. Rhee, Cell Metab. 17 (2013) 73–84. [40] S.H. Chang, J. Garcia, J.A. Melendez, M.S. Kilberg, A. Agarwal, Biochem. J. 371 (2003) 877–885. [41] T. Nguyen, P. Nioi, C.B. Pickett, J. Biol. Chem. 284 (2009) 13291–13295. [42] S.K. Niture, R. Khatri, A.K. Jaiswal, Free Radic. Biol. Med. 66 (2014) 36–44.
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx [43] K. Itoh, K.I. Tong, M. Yamamoto, Free Radic. Biol. Med. 36 (2004) 1208–1213. [44] A. Nishimoto, N. Kugimiya, T. Hosoyama, T. Enoki, T.S. Li, K. Hamano, Int. J. Oncol. 44 (2014) 2077–2084. [45] S.H. Ro, M. Nam, I. Jang, H.W. Park, H. Park, I.A. Semple, M. Kim, J.S. Kim, H. Park, P. Einat, G. Damari, M. Golikov, E. Feinstein, J.H. Lee, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 7849–7854.
11
[46] D. Kukidome, T. Nishikawa, K. Sonoda, K. Imoto, K. Fujisawa, M. Yano, H. Motoshima, T. Taguchi, T. Matsumura, E. Araki, Diabetes 55 (2006) 120–127. [47] M. Juhaszova, D.B. Zorov, S.-H. Kim, S. Pepe, Q. Fu, K.W. Fishbein, B.D. Ziman, S. Wang, K. Ytrehus, C.L. Antos, E.N. Olson, S.J. Sollott, J. Clin. Invest. 113 (2004) 1535–1549. [48] J. Xi, H. Wang, R.A. Mueller, E.A. Norfleet, Z. Xu, Eur. J. Pharmacol. 604 (2009) 111–116.
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
Contents lists available at ScienceDirect
Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity Kyuhwa Seo 1, Sung Hwan Ki 1, Sang Mi Shin ⁎ College of Pharmacy, Chosun University, Gwangju 501-759, South Korea
a r t i c l e
i n f o
Article history: Received 28 January 2015 Accepted 3 March 2015 Available online xxxx Keywords: Glucose deprivation Sestrin2 Reactive oxygen species AMPK Mitochondria Hepatocytes
a b s t r a c t Sestrin2 (SESN2) regulates redox-homeostasis and apoptosis in response to various stresses. Although the antioxidant effects of SESN2 have been well established, the roles of SESN2 in mitochondrial function and metabolic stress have not yet been elucidated. In this study, we investigated the role of SESN2 in mitochondrial dysfunction under glucose deprivation and related signaling mechanisms. Glucose deprivation significantly upregulated SESN2 expression in hepatocyte-derived cells. Antioxidant treatments repressed SESN2 induction under glucose deprivation, this result suggested that reactive oxygen species (ROS) production was involved in SESN2 induction. Moreover, NF-E2-related factor-2 (Nrf2) phosphorylation was accompanied in induction of SESN2 by glucose deprivation. To elucidate the functional role of SESN2, we examined cells that stably overexpressed SESN2. Overexpression of SESN2 inhibited glucose deprivation-induced ROS production and cell death. In addition, under glucose deprivation, the changes in mitochondrial membrane potential, ADP/ATP ratio, and mitochondrial DNA content were significantly restored in SESN2-overexpressing cells. Moreover, siRNA knockdown of SESN2 failed to prevent mitochondrial permeability transition by glucose depletion. Mechanistic investigation showed that glucose deprivation significantly increased AMP-activated protein kinase (AMPK) activation. The recovery of mitochondrial function under glucose deprivation in SESN2-overexpressing cells was not seen in SESN2-overexpressing cells transfected with a dominant-negative AMPK; this result suggested that AMPK activation was responsible for SESN2-mediated mitochondrial protection against glucose deprivation. Treatment with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, an AMPK activator) also provided cytoprotective effects against glucose deprivation. Our findings provide evidence for the functional importance of SESN2–AMPK activation in the protection of mitochondria and cells against glucose deprivation-induced metabolic stress. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The sestrins (SESNs) are evolutionarily conserved stress-inducible genes that are involved in the complex regulation of cell survival in response to various stressful conditions such as genotoxic stress and oxidative stress [1,2]. In mammals, three kinds of SESNs (SESN1–3) have been characterized [2]. SESN1 and SESN2, initially known as PA26 and Hi95, respectively, were identified as p53 target genes and are known to regulate autophagy and cell viability [3,4]. SESN3 was identified as a
Abbreviations: AICAR, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK, AMP-activated protein kinase; AREs, antioxidant response elements; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; FoxOs, forkhead transcription factors; GSK3β, glycogen synthase kinase 3β; HO-1, heme oxygenase-1; mTOR, mammalian target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acetyl cysteine; Nrf2, NF-E2-related factor-2; PRX, peroxiredoxin; ROS, reactive oxygen species; SESN, sestrin; UCP, uncoupling protein ⁎ Corresponding author at: College of Pharmacy, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju 501-759, South Korea. Tel.: +82 62 230 6368; fax: +82 62 222 5414. E-mail address: [email protected] (S.M. Shin). 1 Both contributed equally to this work.
target gene for members of the forkhead transcription factor (FoxO) family and has been reported to affect the induction of SESN1 [5,6]. SESNs modulate redox-homeostasis via the regeneration of peroxiredoxins (PRXs), which controls hydrogen peroxide concentration under the diverse cellular processes [3,7]. Reactive oxygen species (ROS) inactivate PRXs via overoxidation of a catalytic cysteine residue. To sequester excessive ROS, PRXs must be restored via the sulfinyl reductase system. SESNs have been reported to play a critical role in the inhibition of intracellular ROS by rescuing of PRX from inactivation [7]. Recently, Shin et al. directly showed that SESN2 plays a critical role in cytoprotection under hydrogen peroxide-induced oxidative stress [8]. SESNs can also affect cell growth by controlling of mammalian target of rapamycin (mTOR) activity, but this function is not mediated by the modulation of redox activity [1,9]. mTOR is a master kinase complex that regulates protein synthesis, cell growth and metabolism. SESNs negatively regulate mTOR activity by AMP-activated protein kinase (AMPK) activation and TSC2 phosphorylation under genotoxic stress [1]. In addition, SESNs interact directly with AMPK in response to metabolic stress, thus helping to protect cells against stress-induced apoptosis [9]. In general, AMPK is activated by stimuli that increase the
http://dx.doi.org/10.1016/j.cellsig.2015.03.003 0898-6568/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
2
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
intracellular AMP/ATP ratio, and it inhibits energy-consuming processes and stimulates ATP-generating pathways in order to restore the energy balance [10,11]. Therefore, SESNs-mediated AMPK signaling has been proposed to play a crucial role in the regulation of cellular energy homeostasis and metabolism [2,12]. AMPK is activated in response to metabolic stress and plays a role in compensatory responses that protect cells from cell death by restoring mitochondrial function [11]. In our previous study, we reported that various AMPK activators such as resveratrol and 5-aminoimidazole-4carboxamide-1-β-D-ribofuranoside (AICAR) could protect cells from mitochondrial dysfunction-mediated apoptosis in hepatocytes [13,14]. Many reports have provided evidence that mitochondria are deeply involved in the regulation of metabolic stress-induced cell death [15]. Mitochondria are responsible for not only for ATP production, but also for the initiation of apoptosis [15]. The various cellular events in apoptosis such as loss of mitochondrial membrane potential and the subsequent release of cytochrome c are mediated in mitochondria [15]. Metabolic stress such as a decrease in the glucose supply results in the depletion of intracellular ATP level and sensitizes cells to apoptosis [16]. However, it is unclear whether SESNs, as upstream regulators of AMPK, could exert protective effects on mitochondria and cells. Metabolic stress changes electron transports in mitochondria and provokes the release of ROS [17,18]. Therefore, it has been reported to increase the expression of markers that indicate oxidative stress in cells [17,19]. Moreover, many enzymes responsible for redox signaling are induced as a major defense system against oxidative stress [20]. Heme oxygenase-1 (HO-1), an antioxidant gene whose expression is mediated by NF-E2-related factor-2 (Nrf2) and which is induced by glucose deprivation, has been shown to increase cell viability via the reduction of ROS production [21]. However, the possibility whether SESNs has protective effects against metabolic stress-induced apoptosis has not been reported. Based on the previous reports that SESN2 is an Nrf2-mediated antioxidant enzyme and activates AMPK, in this study, we investigated the role of SESN2–AMPK signaling in glucose depletion-induced apoptosis. We found that SESN2–AMPK signaling could exert a protective effect against glucose deprivation-induced cell death and that this effect is mediated by restoration of mitochondrial function. 2. Materials and methods 2.1. Materials Anti-SESN2 antibody was purchased from Proteintech (Chicago, IL). Anti-Nrf2 antibody, anti-PARP antibody, anti-Bcl − xL antibody, and rhodamine 123 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mn-TBAP was purchased from Cayman Chemical (Ann Arbor, MI). Cyclosporin A was purchased from Calbiochem (San Diego, CA). Caspase-3 antibody, p-ACC antibody, p-AMPK antibody, and AMPKα antibody were purchased from Cell Signaling (Danvers, MA). p-Nrf2 antibody was purchased from NOVUS Biologicals (Littleton, CO). Glucosefree Dulbecco's modified Eagle's medium (DMEM) was obtained from Life Technology (Gaithersburg, MD). Dimethylsulfoxide (DMSO), trolox, PEG-catalase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), anti-βactin antibody, rotenone, AICAR, N-acetyl cysteine (NAC) and other reagents were purchased from Sigma Chemicals (St. Louis, MO).
were maintained in media containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 50 units/mL penicillin and 50 μg/mL streptomycin, and cultured at 37 °C in a humidified atmosphere with 5% CO2. For experiments, cells were plated in plates for 2–3 days (i.e., 80% confluency) and serum starved overnight before treatments. 2.3. Establishment of a stable cell line expressing SESN2 Cells stably expressing SESN2 were established as previously described [22]. HepG2 cells were transfected with the plasmid pCMVTag3A (mock-transfected) or pCMV-SESN2 by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 1 day of transfection, colonies of geneticin-resistant cells were selected by treatment with geneticin (500 μg/mL) and then amplified in culture. SESN2 overexpression was established by immunoblotting. 2.4. Primary hepatocyte isolation Animal experiments were conducted under the guidelines of the Institutional Animal Use and Care Committee at Chosun University. Primary hepatocytes were isolated from male ICR mice (Samtako, Korea) according to standard procedures as follows [23]. Briefly, the mouse was injected with Zoletil (Virbac, France) for anesthesia and cannulated portal vein to perfuse the liver by HBSS containing 0.1% collagenase and calcium. After the liver was collected, it was minced gently with scissors. The minced tissue was suspended in DMEM, filtered through a cell strainer, and then centrifuged at 400 rpm for 5 min to separate parenchymal and nonparenchymal cells. Isolated hepatocytes were seeded on collagen-coated dishes and cultured in DMEM containing 75 units/mL penicillin and 75 μg/mL streptomycin with 10% FBS. Cell viability was determined via trypan blue staining; the viability of the isolated hepatocytes was usually 80–90%. 2.5. MTT assay Cells were seeded in a 48-well plate. For the assay, an MTT solution in PBS was added to each well, and the cells were incubated for 3 h. The resulting formazans were solubilized in DMSO, and their absorbance was measured at 570 nm using a microplate reader (Spectramax 190, Molecular Device, Sunnyvale, CA). Cell viability was calculated relative to the untreated control according to the following formula: viability (% control) = 100 × (absorbance of treated sample) / (absorbance of control). 2.6. Measurement of ROS generation The level of hydrogen peroxide production was determined by measuring the increase in dichlorofluorescein fluorescence after treatment with DCFH-DA, a cell-permeable nonfluorescent probe that is cleaved by intracellular esterases and oxidized primarily by hydrogen peroxide. Cells were stained with 10 μM DCFH-DA during the final hour of incubation. The cells were then harvested by trypsinization and washed twice with PBS. The intensity of the fluorescence in the cells was measured using a fluorescence microplate reader (Gemini XPS, Molecular Device, Sunnyvale, CA). ROS production was normalized to the protein concentration in each treated sample and calculated relative to the vehicletreated control.
2.2. Cell culture 2.7. Mitochondrial membrane permeability analysis The HepG2 and AML12 cell lines were purchased from the American Type Culture Collection (Manassas, VA). HepG2 cells were maintained in DMEM and Huh7 cells were maintained in RPMI. AML12 cells were cultured in 1:1 mixture of DMEM and Ham's F12 medium (Hyclone, Logan, UT) with 0.005 mg/mL insulin, 0.005 mg/mL transferrin, and 5 ng/mL selenium (Life Technologies, Gaithersburg, MD). All cells
Changes in mitochondrial membrane permeability were determined by staining with rhodamine 123, a membrane-permeable cationic fluorescent dye. Cells were treated with 0.05 μg/mL rhodamine 123 for last 1 h treatment. Sample collection and detection of fluorescence intensity were conducted as described in ROS production.
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
3
2.8. Immunoblot analysis
2.13. siRNA knockdown experiment
Cell lysates were prepared according to previously published procedures [13] and resolved using 7.5% or 12% gel electrophoresis. The proteins were then electophoretically transferred to nitrocellulose membranes. After the membranes were blocked, they were incubated with primary antibody at 4 °C overnight and then incubated with a secondary antibody. Protein bands of interest were identified using an ECL chemiluminescence system (GE Healthcare, Buckinghamshire, UK). Immunoblotting for β-actin confirmed equal loading of proteins.
Cells were transfected with either an siRNA directed against human SESN2 (Cat No. L-019134-02-0005, ON-TARGETplus SMARTpool; Dharmacon Inc., Lafayette, CO) or a non-targeting control siRNA (100 pmol/mL) using Lipofectamine 2000 (Invitrogen, San Diego, CA). After transfection for 24 h, cells were incubated in glucose (5.6 mM) or glucose-free DMEM. The knockdown of SESN2 was confirmed by immunoblot analysis. 2.14. Statistical analysis
2.9. RNA isolation and RT-PCR analysis RNA isolation was performed using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The RNA was then reverse transcribed using an oligo(dT)16 primer. PCR was conducted using a PCR premix (Bioneer, Daejeon, Korea) and a thermal cycler (Bio-Rad, Hercules, CA). GAPDH was used as a control for the amount of total mRNA. The following primer pairs were used for PCR: human SESN1 5′-CTTCTGGAGGCAGTTCAAGC-3′ (forward) and 5′-TGAATGGCAGCC TGTCTTCAC-3′ (reverse); human SESN2 5′-CTCACACCATTAAGCATG GAG-3′ (forward) and 5′-CAAGCTCGGAATTAATGTGCC-3′ (reverse); and human GAPDH 5′-GAAGATGGTGATGGGATTTC-3′ (forward) and 5′-GAAGGTGAAGGTCGGAGTC-3′ (reverse). 2.10. Plasmid transfection and luciferase assay A human SESN2 promoter-driven luciferase construct and dominant negative form of AMPK (D157A; DN-AMPK) were prepared as previously described [8,24]. Cells were plated in 12-well plates, incubated overnight, and then serum starved for 6 h. Transient transfection with the promoter-luciferase construct, pRL-TK plasmid (a plasmid that encodes for Renilla luciferase and is used to normalize transfection efficacy), and either DN-AMPK or pCDNA (control) was performed for 3 h using Lipofectamine (Invitrogen, San Diego, CA). The transfected cells were then incubated in minimum essential media containing 1% FBS for 14 h. Then, the luciferase activity in the lysates was determined using the dual-luciferase reporter assay system (Promega, Madison, WI) according to previously published procedures [8]. 2.11. Measurement of ADP/ATP ratio The ADP/ATP ratio was assessed using the EnzyLight ADP/ATP ratio assay kit (BioAssay Systems, Hayward, CA) according to the manufacturer's instructions. Briefly, the ATP reagent from the kit was added to cells grown in a 96-well plate. Then, luminescence due to ATP (RLU A) was measured using a luminometer (Promega, Madison, WI), and background luminescence (RLU B) was measured 10 min later. Next, ADP reagent was added to the cells and luminescence due to ADP (RLU C) was measured. Finally, the ADP/ATP was calculated by subtracting RLU B from RLU C and then dividing the result by RLU A. 2.12. Measurements of mitochondrial DNA Total DNA was extracted from cells according to the manufacturer's instructions (Nucleogen, Siheung, Korea). Levels of cytochrome c oxidase subunit II (mtCOX II) transcribed from mitochondrial DNA (mtDNA) were quantified by real-time PCR and normalized using nuclear-encoded receptor-interacting protein 140 (RIP140). The following primer pairs were used: human mtCOX II 5′-ACCTGCGACTCC TTGACGTTG-3′ (forward) and 5′-TAGGACGATGGGCATGAAACTG-3′ (reverse), and human RIP140 5′-GCTGGGCATAATGAAGAGGA-3′ (forward) and 5′-CAAAGAGGCCAGTAATGTGCTATC-3′ (reverse). Realtime PCR was performed using StepOne (Applied Biosystems, Foster City, CA) with a SYBR Green premix according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).
For each statistically significant effect of treatment, one-way analysis of variance (ANOVA) was used for comparisons between multiple group means. The data were expressed as means ± S.E. from at least three independent experiments. The criterion for statistical significance was set at p b 0.05 or p b 0.01. 3. Results 3.1. Glucose levels regulates SESN2 expression in hepatocytes SESN2 is an antioxidant enzyme that also regulates AMPK in response to various stresses [1,25]. Given that AMPK is a cellular energy sensor, we first examined whether SESN2 is regulated by glucose levels in hepatocytes. When we compared the level of SESN2 expression in primary hepatocytes isolated from mice that had been fasted for 18 h with that in hepatocytes isolated from nonfasted mice, we found that the level of SESN2 expression was higher in the hepatocytes from fasted mice (Fig. 1A, left). In hepatocyte-derived cell lines (AML12 and Huh7 cells), SESN2 induction in response to glucose deprivation was also observed (Fig. 1A, right). To further investigate the effect of glucose levels on SESN2 induction, HepG2 cells were incubated in media containing various concentrations (0–25 mM) of glucose, and then levels of SESN2 mRNA and protein expression were measured. When the cells were incubated in glucose-free media, levels of SESN2 mRNA and protein expression were increased (Fig. 1B and C). However, incubation of the cells in media containing high glucose (25 mM) did not significantly affect SESN2 expression when compared with normal glucose (5.6 mM). Moreover, the level of SESN1 mRNA expression remained unchanged (Fig. 1B). Incubation of cells with glucose-free media resulted in a marked increase in SESN2 mRNA expression starting from 6 h after the beginning of the experiment and peaking at 12 h, but the expression of SESN1 was not changed (Fig. 1D). A significant increase in SESN2 protein expression in cells incubated in glucose-free media was detected from 12 h after the start of the experiment and peaked at 18 h (Fig. 1E). To confirm that SESN2 was induced in response to glucose deprivation, cells were transiently transfected with a human SESN2 promoter-driven luciferase construct and then incubated in glucosefree medium. Glucose deprivation significantly increased the activity of the reporter construct; this result indicates that transcriptional induction of SESN2 was upregulated (Fig. 1F). Collectively, these results suggest that glucose deprivation increases the SESN2 induction. 3.2. Involvement of ROS production in glucose deprivation-induced SESN2 upregulation Previously, we found that SESN2 is induced by oxidative stress and knockdown of SESN2 promoted cell death mediated by hydrogen peroxide [8]. In addition, SESN2 has been shown to have cytoprotective activity against oxidative stress in various tissues [22,26,27]. Therefore, we investigated whether overproduction of ROS under glucose deprivation could lead to the induction of SESN2. First, we measured intracellular ROS accumulation by using DCFH-DA. When cells were incubated in glucose-free medium for 12 h, a significant increase in intracellular ROS levels was observed (Fig. 2A). To confirm that ROS induced SESN2
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
4
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
Fig. 1. Effect of glucose levels on Sestrin2 (SESN2) expression in hepatocytes-derived cells. (A) Immunoblot analysis. SESN2 protein level was determined by using lysates from primary hepatocytes from mice that had fasted for 18 h (left). AML12 and Huh7 cells were incubated in glucose-containing (5.6 mM) or glucose-free DMEM for 18 h (right). Lysates from these cells were then used to determine SESN2 protein level via immunoblotting. The blots shown are representative of data from at least 3 different replicates. (B) RT-PCR assays. HepG2 cells were incubated in the indicated concentrations of glucose for 6 h, and then the SESN1 or SESN2 transcripts were analyzed by RT-PCR. The results shown are representative of data from at least 3 different replicates. (C) Immunoblot analysis. HepG2 cells were incubated in the indicated concentrations of glucose for 12 h. Lysates from these cells were then used to determine SESN2 protein level via immunoblotting. The blots shown are representative of data from at least 3 different replicates. (D) RT-PCR assays. HepG2 cells were incubated in glucose-free DMEM for the indicated time period (0−12 h). SESN1 or SESN2 transcripts were then analyzed by RT-PCR. The results shown are representative of data from at least 3 different replicates; ⁎p b 0.05 or ⁎⁎p b 0.01 when compared to the control. (E) Immunoblot analysis. The lysates of cells incubated in glucose-free DMEM for 0−18 h were immunoblotted for SESN2 protein level. The blots shown are representative of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the control. (F) Luciferase activity. SESN2 luciferase activity was determined from the cell lysates incubated in glucose-free DMEM for 12 h. Data were expressed as the mean ± S.E. from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control.
expression, various antioxidants were added to the glucose-free medium. Treatment of the cells with NAC markedly inhibited the induction of SESN2 expression by glucose deprivation (Fig. 2B). In hepatocytes, mitochondria are the major sites for ROS production [28]. Hence, the
superoxide dismutase (SOD) mimetic Mn-TBAP was used to scavenge mitochondria-derived free radicals. The induction of SESN2 by glucose deprivation was suppressed by Mn-TBAP (Fig. 2C, left). Moreover, a low concentration of rotenone, a mitochondrial complex I inhibitor
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
5
Fig. 2. Involvement of ROS in glucose deprivation-induced SESN2 induction. (A) Measurement of ROS production. Cells were incubated in glucose (5.6 mM) or glucose-free DMEM for 12 h. ROS production was assessed by DCF fluorescent intensity. The data are expressed as mean ± S.E. from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control. (B) Immunoblot analysis. Cells were incubated with or without NAC (5 mM) in glucose (5.6 mM) or glucose-free DMEM for 12 h. Then, lysates from these cells were used to determine SESN2 protein level via immunoblotting. The blots shown are representative of data from at least 3 different replicates; ⁎p b 0.05 when compared to the glucose-incubated control; #p b 0.05 when compared to cells incubated in glucose-free DMEM without NAC. (C) Immunoblot analysis. Cells were incubated with or without Mn-TBAP (20 μM, left) or rotenone (10 nM, right) in glucose (5.6 mM) or glucose-free DMEM for 12 h. Then, lysates from these cells were used to determine SESN2 protein level via immunoblotting. The blots shown are representative of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; #p b 0.05 or ##p b 0.01 when compared to cells incubated in glucose-free DMEM without Mn-TBAP or rotenone.
which reduces ROS levels through inhibiting the mitochondrial respiratory chain [29], inhibited the induction of SESN2 by glucose deprivation (Fig. 2C, right); this result indicates that mitochondria-derived ROS are involved in the induction of SESN2 by glucose deprivation. Previously, we demonstrated that ROS-mediated SESN2 expression is mediated with Nrf2-antioxidant response element (ARE) activation [8]. In addition, Lee et al. showed that glucose deprivation increased nuclear translocation of Nrf2 and Nrf2–ARE-DNA binding in HepG2 cells [21]. To elucidate whether the induction of SESN2 by glucose deprivation was accompanied by Nrf2 activation, the phosphorylation of Nrf2 was observed. Incubation of cells in glucose-free medium increased the phosphorylation of Nrf2 (Fig. 3A). We also examined the functional role of ARE in SESN2 gene induction by using a ΔARESESN2 promoter. Specific disruption of the ARE in the promoter region
of the SESN2 gene significantly decreased the induction of SESN2 by glucose deprivation (Fig. 3B). These observations indicate that the Nrf2–ARE system contributes to the induction of SESN2 by glucose deprivation. Taken together, these results suggest that glucose deprivation produces ROS, which in turn upregulates SESN2 expression via Nrf2–ARE activation. 3.3. Involvement of mitochondrial damage in glucose deprivation-induced apoptosis In HepG2 cells, glucose deprivation significantly increases apoptotic cell death [21]. Since glucose deprivation leads to produce mitochondrial ROS and deplete intracellular ATP, glucose-depleted cells cannot maintain mitochondrial membrane permeability and the loss of mitochondrial
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
6
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
Fig. 3. Involvement of Nrf2 activation in glucose deprivation-induced SESN2 induction. (A) Immunoblot analysis. The lysates of cells incubated in glucose-free DMEM for 0−6 h were immunoblotted. The blots shown are representative of data from at least 3 different replicates; ⁎p b 0.05 when compared to the control. (B) Luciferase activity. Cells had been transfected with pGL4-phSESN2 or pGL3-phSESN2-ΔARE and incubated in glucose (5.6 mM) or glucose-free DMEM for 12 h. Luciferase activity was determined from the cell lysates. Data were expressed as the mean ± S.E. from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control in pGL4-phSESN2 transfected cells; ##p b 0.01 when compared to cells incubated in glucose-free DMEM after pGL4-phSESN2 transfection.
membrane permeability activates the apoptotic signal cascade [16,30]. Therefore, in an attempt to correlate mitochondrial damage with glucose deprivation-induced apoptosis, mitochondrial membrane permeability was measured using rhodamine 123 staining [31]. When cells were incubated in glucose-free medium, the intensity of rhodamine 123 fluorescence significantly decreased; this result indicates that glucose deprivation induces mitochondrial membrane damage (Fig. 4A). In accordance with previous reports that cyclosporin A prevents mitochondrial damage by inhibiting mitochondrial membrane transition pore formation [14,32,33], the addition of cyclosporin A was found to restore the loss of membrane permeability induced by glucose deprivation (Fig. 4A). In addition, cyclosporin A significantly prevented glucose deprivation-induced
Fig. 4. Involvement of mitochondrial dysfunction in glucose deprivation-induced apoptosis. (A) Measurement of mitochondrial membrane permeability changes. Cells were incubated in the indicated DMEM with or without cyclosporin A (10 μg/mL) for 18 h. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; #p b 0.05 when compared to cells incubated in glucose-free DMEM without cyclosporin A. (B) Cell viability assay. Cells were incubated in indicated DMEM without or with cyclosporin A (10 μg/mL) for 24 h. The effect of cyclosporin A on cell viability was then assessed using MTT assay. Data are expressed as the means ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; ##p b 0.01 when compared to cells incubated in glucose-free DMEM without cyclosporin A. (C) Cell viability assay. Cells were incubated in indicated DMEM with or without trolox (100 μM), PEG-catalase (catalase, 1000 U/mL), or Mn-TBAP (20 μM) for 24 h. The effect of these antioxidants on cell viability was then assessed using the MTT assay. The data are expressed as the means ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; ##p b 0.01 when comparing treatment groups under glucose-free DMEM incubation.
cell death (Fig. 4B). Moreover, trolox (vitamin E analog and hydroxyl radical scavenger), catalase (a hydrogen peroxide scavenger), and Mn-TBAP also prevented glucose deprivation-induced cell death from glucose
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
7
deprivation (Fig. 4C); this result suggests that intracellular ROS, including mitochondrial ROS, play an important role in apoptosis. Taken together, these results provide evidence that glucose deprivation-induced ROS cause mitochondrial damage, which subsequently lead to cell death. 3.4. Prevention of glucose deprivation-induced cytotoxicity by SESN2 overexpression To determine whether SESN2 has a cytoprotective effects, we prepared cell lines that stably overexpressed SESN2; SESN2 overexpression was confirmed by western blotting (Fig. 5A, upper). In comparison to the cells which had been stably transfected with pCMV-Tag3A (mocktransfected cells), SESN2 overexpression repressed glucose deprivationinduced cell death (Fig. 5A, lower). The protective effects of SESN2 were confirmed by observing proteins associated with apoptosis. PARP cleavage, caspase-3 activation, and a decrease in Bcl − xL expression, all of which were induced by glucose deprivation, did not occur in SESN2overexpressing cells (Fig. 5B). Moreover, glucose deprivation-induced ROS production was reduced in SESN2-overexpressing cells (Fig. 5C). Overall, these results indicate that SESN2 protects cells against glucose deprivation-induced apoptosis by reducing ROS production. 3.5. Inhibition of glucose deprivation-induced mitochondrial damage by SESN2 overexpression Next, we determined whether SESN2 could affect mitochondrial function under glucose deprivation. Since mitochondria play a central role in intracellular ATP production, the ADP/ATP ratio was used as a measure of mitochondrial function. ADP/ATP ratio was increased in mock-transfected cells by glucose deprivation. In contrast, the ADP/ ATP ratio of SESN2-overexpressing cells was decreased when compared with mock-transfected cells (Fig. 6A). In addition, glucose deprivation in mock-transfected cells significantly decreased levels of mtDNA, which represents numbers of mitochondria, but mtDNA levels were restored in SESN2-overexpressing cells (Fig. 6B). Moreover, rhodamine 123 fluorescence intensity under glucose deprivation was significantly higher in SESN2-overexpressing cells than in mock-transfected cells; this result suggests that SESN2 helps to preserve the mitochondrial membrane potential (Fig. 6C). Similarly, transfection with an siRNA directed against human SESN2 potentiated the glucose deprivation-induced loss of mitochondrial permeability (Fig. 6D, lower); SESN2 knockdown was confirmed by immunoblot analysis (Fig. 6D, upper). These results indicate that SESN2 plays a critical role in the maintenance of mitochondrial function under glucose deprivation-induced oxidative stress. 3.6. The role of AMPK activation in the recovery of mitochondrial function Previous studies have shown that SESN2 regulates AMPK activity, which activates an adaptive response during various stresses [1,9,25]. In view of the fact that AMPK is a sensor of energy status, we examined the role of the SESN2–AMPK axis in protecting mitochondria during glucose deprivation. Glucose deprivation increased phosphorylation of ACC, which represents cellular AMPK activity (Fig. 7A). The increase of ACC phosphorylation by glucose deprivation in SESN2-overexpressing cells was similar to mock-transfected cells (Fig. 7B). Next, we investigated the role of AMPK activation in SESN2 mediated-protective effects in mitochondria. The recovery of fluorescence intensity elicited by SESN2 overexpression was significantly reversed by DN-AMPK overexpression (Fig. 7C, lower). Overexpression of the DN-AMPK was verified by observation of AICAR-induced ACC phosphorylation (Fig. 7C, upper). To assess whether AMPK activation could protect cells against glucose deprivation, an MTT assay was conducted on cells treated with AICAR, an AMPK activator. AICAR treatment resulted in a notable increase in cell viability in glucose-deprived cells (Fig. 7D). These results strongly support the hypothesis that the protection provided by SESN2 against
Fig. 5. Inhibition of glucose deprivation-induced apoptosis by SESN2 overexpression. (A) Cell viability assay. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 24 h. Cell death was determined by the MTT assay. Data represent the mean ± S.E. of at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucoseincubated mock-transfected cells; ##p b 0.01 when comparing mock-transfected cells and SESN2-overexpressing cells in glucose free-DMEM. (B) Immunoblot analysis. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 24 h. Then, lysates from these cells were used to determine levels of apoptotic proteins via immunoblotting. The blots shown are representative of data from at least 3 different replicates. (C) Measurement of ROS generation. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 12 h. ROS levels were then assessed via DCF fluorescent intensity. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated mock-transfected cells; #p b 0.05 when comparing mock-transfected cells in glucose-free DMEM and SESN2-overexpressing cells in glucose free-DMEM.
glucose deprivation-induced mitochondria damage and cell death might be linked to AMPK activation. 4. Discussion In the present study, we show that increased ROS production caused by glucose deprivation induces SESN2, which protects mitochondria
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
8
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
Fig. 6. Inhibition of mitochondrial dysfunction by SESN2 overexpression. (A) ADP/ATP ratio assay. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 15 h. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎p b 0.05 when compared to glucose-incubated mock-transfected cells; ##p b 0.01 when comparing mocktransfected cells and SESN2-overexpressing cells in glucose free-DMEM. (B) mtDNA contents. Cells were incubated in DMEM with glucose (5.6 mM) or glucose-free DMEM for 15 h. The samples were then subjected to real-time PCR analysis with primers for the mtDNA region COXII. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated mock-transfected cells; ##p b 0.01 when comparing mock-transfected cells and SESN2-overexpressing cells in glucose free-DMEM. (C) Measurement of mitochondrial membrane permeability changes. Cells were incubated in DMEM containing glucose (5.6 mM) or glucose-free DMEM for 18 h. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated mock-transfected cells; ##p b 0.01 when comparing mock-transfected cells and SESN2-overexpressing cells in glucose free-DMEM. (D) Measurement of mitochondrial membrane permeability changes. Cells were transfected with control siRNA (siRNA CON) or SESN2 siRNA (siRNA SESN2). Then, cells were incubated in DMEM containing glucose (5.6 mM) or glucose-free DMEM for 12 h; ⁎p b 0.05 when compared to glucose-incubated control siRNA-transfected cells; #p b 0.05 when comparing control siRNA-transfected cells and SESN2 siRNA-transfected cells in glucose free-DMEM.
and cells from metabolic stress. In addition, our results suggest that SESN2–AMPK activation under energy depletion plays a crucial role in the adaptive survival response. The beneficial roles of SESNs are well documented. p53-Mediated SESN1 and SESN2 induction inhibits mTOR signaling via AMPK activation and TSC phosphorylation, and thus leads to metabolic arrest [1,9, 34]. SESNs have been demonstrated to induce autophagy, which leads to protect neuron and renal tubules against cytoxicity [26,35]. In addition, SESN2 attenuates high glucose-induced dysfunction of endothelial nitric oxide synthase and synthesis of fibronectin in glomerular mesangial cells [36]. Loss of epithelial barrier integrity and muscle degeneration is also prevented by SESN2 induction [37,38]. Moreover, SESN2 is involved in lipid lowering effects by downregulation of liver X receptor-α-dependent lipogenic gene expression [23]. Our study shows that SESN2 has an additional, crucial role in mitochondrial protection under hyponutrition. Collectively, SESNs have been demonstrated to show beneficial effects against oxidative injury, lipid accumulation, and tissue degeneration.
Three isoforms of SESNs (SESN1–3) have been reported in mammals. Although all three isoforms have been reported to decrease intracellular ROS, the induction of each SESN is regulated by different mechanisms. In general, SESN1 and SESN2 are induced by various stresses, which are mediated with p53 activation. SESN3 expression is regulated by Akt and FoxO-mediated signaling, but not induced by treatment with 2-deoxyglucose (an inhibitor of glycolysis) [9]. Here, we show that expression of SESN2 but not SESN1 is induced by glucose deprivation in HepG2 cells (Fig. 1). Bae et al., also observed that fasting and refeeding states regulate SESN2 level in mouse liver [39]. Moreover, we also observe that ROS play a critical role in SESN2 induction under glucose deprivation (Fig. 2). In accordance with present study, previous study reported that tert-butyl hydroquinone caused SESN2 induction but not SESN1 or SESN3 in HepG2 cells [8]. Collectively, these results indicate that among three isoforms of SESNs, SESN2 is sensitively induced under oxidative and metabolic stress in hepatocytes-derived cells. In a report from Ahmad et al., glucose depletion increases representative markers of oxidative stress [17]. Indeed, cellular antioxidants
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
9
Fig. 7. Involvement of AMP-activated protein kinase (AMPK) activation in SESN2-mediated mitochondrial protection. (A) Immunoblot analysis. The lysates of cells that had been incubated in glucose-free DMEM for 0–18 h were immunoblotted. The blots shown are representative of data from at least 3 different replicates; ⁎p b 0.05 or ⁎⁎p b 0.01 when compared to the control. (B) Immunoblot analysis. Cells were incubated in DMEM containing glucose (5.6 mM) or glucose-free DMEM for 12 h. Protein levels in lysates from these cells were then determined via immunoblotting. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated each control cells. (C) Measurement of mitochondrial membrane permeability changes. Mock-transfected or SESN2-overexpressing cells were transfected with a construct expressing a dominant-negative form of AMPK (DN-AMPK) or pCDNA (i.e., empty plasmid). Then, cells were incubated in DMEM containing glucose (5.6 mM) or glucose-free DMEM for 12 h. Data represent the mean ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to glucose-incubated pCDNA and mock-transfected cells; ##p b 0.01 when comparing pCDNA and mock-transfected cells and pCDNA transfected SESN2 overexpressing cells in glucose free-DMEM. (D) Cell viability assay. Cells were incubated with or without AICAR (2 mM) in indicated DMEM for 24 h. The effect of AICAR on cell viability was then assessed using the MTT assay. The data were expressed as means ± S.E. of data from at least 3 different replicates; ⁎⁎p b 0.01 when compared to the glucose-incubated control; ##p b 0.01 when compared to cells incubated in glucose-free DMEM without AICAR.
including HO-1 and catalase are induced by exposure to glucose-free medium [21,40]. This induction is accompanied by an increase in ROS production [21,40]. It is well established that ROS induce a large number of antioxidant genes by activation of ARE and transcriptional modulation of genes [8,21,41]. Nrf2 is the transcription factor that activates ARE-mediated antioxidant gene expression [41]. Previous study by Lee
et al. reported that nuclear translocation of Nrf2 and Nrf2–ARE-DNA binding were increased by glucose deprivation, which contributed to HO-1 induction [21]. Since translocation or stabilization of Nrf2 is regulated by phosphoylation [42], we observe Nrf2 phosphorylation under glucose deprivation (Fig. 3A). The phosphorylation of Nrf2 Ser40 has been reported to result in the escape or release of Nrf2 from Kelch-like
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
10
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx
mitochondrial dysfunction and cell death (Fig. 8). These findings provide novel insights into the mechanisms underlying metabolic stress-mediated cellular signaling and into the importance of SESN2 in the cellular adaptive response. Acknowledgment This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No. R13-2008-010-00000-0). References
Fig. 8. Schematic diagram illustrating that SESN2 inhibits mitochondrial dysfunction and apoptosis against glucose deprivation-induced stress by AMPK activation.
ECH-associated protein 1, an inhibitory regulator of Nrf2 [43]. Therefore, increase of Nrf2 Ser40 phosphorylation by glucose deprivation represents Nrf2 activation. In addition, we show that SESN2 induction is mediated with Nrf2–ARE activation as evidenced by SESN2 luciferase activity containing ARE or not (Fig. 3B). Another study by Ben-Sahra et al. demonstrated that ATP depletion upregulated SESN2 level in an Akt-dependent but p53-independent manner [9]. In view of the fact that hypoxic conditions induce SESN2 [3], glucose deprivation-induced hypoxia-inducible factor-1α might contribute to upregulate SESN2 [44]. Further detailed study is needed to understand the mechanisms underlying the transcriptional regulation of SESN2 in response to metabolic stress. The evidence indicating a direct link between SESN2 and mitochondria remains questionable. In adipose tissues, the antioxidant activity of SESN2 contributes to inhibit mitochondrial uncoupling protein (UCP) expression and plays a critical role in fat metabolism [45]. Here, we show that the protective effects on mitochondria by SESN2 require AMPK activation. Therefore, downstream targets of AMPK probably play a crucial role in SESN2-mediated mitochondrial protection. The primary regulator of AMPK activity is the cellular AMP/ATP ratio [10]. In response to an increase in the AMP/ATP ratio, AMPK represses ATPconsuming processes and increases ATP synthesis [11]. Since mitochondria are the sites of ATP production, mitochondria may be target of AMPK to preserve cellular energy content [11]. It is well accepted that AMPK suppresses ROS accumulation in mitochondria by increasing the levels of mRNA expression for peroxisome proliferators-activated response-coactivator-1, UCP, and MnSOD [46]. AMPK activation is also able to regulate mitochondrial permeability by inhibiting glycogen synthase kinase 3β (GSK3β). GSK3β activated by oxidative stress can translocate into the mitochondria, where it phosphorylate components of the permeability transition pore and induces the collapse of membrane potential [47,48]. Therefore, the inhibitory phosphorylation of GSK3β by AMPK contributes to mitochondrial protection. Moreover, because both SESN2 and AMPK negatively regulate mTOR activity, ATP-consuming processes by mTOR can be reduced to help maintain ATP levels under metabolic stress. 5. Conclusion All together, we have shown that SESN2–AMPK activation inhibits glucose deprivation-induced ROS production, thereby preventing
[1] A.V. Budanov, M. Karin, Cell 134 (2008) 451–460. [2] A.V. Budanov, J.H. Lee, M. Karin, EMBO Mol. Med. 2 (2010) 388–400. [3] A.V. Budanov, T. Shoshani, A. Faerman, E. Zelin, I. Kamer, H. Kalinski, S. Gorodin, A. Fishman, A. Chajut, P. Einat, R. Skaliter, A.V. Gudkov, P.M. Chumakov, E. Feinstein, Oncogene 21 (2002) 6017–6031. [4] M.C. Maiuri, S.A. Malik, E. Morselli, O. Kepp, A. Criollo, P.-L. Mouchel, R. Carnuccio, G. Kroemer, Cell Cycle 8 (2009) 1571–1576. [5] V. Nogueira, Y. Park, C.-C. Chen, P.-Z. Xu, M.-L. Chen, I. Tonic, T. Unterman, N. Hay, Cancer Cell 14 (2008) 458–470. [6] H. Tran, A. Brunet, J.M. Grenier, S.R. Datta, A.J. Fornace, P.S. DiStefano, L.W. Chiang, M.E. Greenberg, Science 296 (2002) 530–534. [7] A.V. Budanov, A.A. Sablina, E. Feinstein, E.V. Koonin, P.M. Chumakov, Science 304 (2004) 596–600. [8] B.Y. Shin, S.H. Jin, I.J. Cho, S.H. Ki, Free Radic. Biol. Med. 53 (2012) 834–841. [9] I. Ben-Sahra, B. Dirat, K. Laurent, A. Puissant, P. Auberger, A. Budanov, J.F. Tanti, F. Bost, Cell Death Differ. 20 (2013) 611–619. [10] D.G. Hardie, Nat. Rev. Mol. Cell Biol. 8 (2007) 774–785. [11] G.R. Steinberg, B.E. Kemp, Physiol. Rev. 89 (2009) 1025–1078. [12] J.H. Lee, A.V. Budanov, S. Talukdar, E.J. Park, H.L. Park, H.-W. Park, G. Bandyopadhyay, N. Li, M. Aghajan, I. Jang, A.M. Wolfe, G.A. Perkins, M.H. Ellisman, E. Bier, M. Scadeng, M. Foretz, B. Viollet, J. Olefsky, M. Karin, Cell Metab. 16 (2012) 311–321. [13] S.M. Shin, I.J. Cho, S.G. Kim, Mol. Pharmacol. 76 (2009) 884–895. [14] S.M. Shin, S.G. Kim, Mol. Pharmacol. 75 (2009) 242–253. [15] D.R. Green, J.C. Reed, Science 281 (1998) 1309–1312. [16] K.H. Moley, M.M. Mueckler, Apoptosis 5 (2000) 99–105. [17] I.M. Ahmad, N. Aykin-Burns, J.E. Sim, S.A. Walsh, R. Higashikubo, G.R. Buettner, S. Venkataraman, M.A. Mackey, S.W. Flanagan, L.W. Oberley, D.R. Spitz, J. Biol. Chem. 280 (2005) 4254–4263. [18] E. Cadenas, K.J.A. Davies, Free Radic. Biol. Med. 29 (2000) 222–230. [19] J.D. Browning, J.D. Horton, J. Clin. Invest. 114 (2004) 147–152. [20] J.L. Evans, I.D. Goldfine, B.A. Maddux, G.M. Grodsky, Endocr. Rev. 23 (2002) 599–622. [21] H.G. Lee, M.H. Li, E.J. Joung, H.K. Na, Y.N. Cha, Y.J. Surh, Antioxid. Redox Signal. 13 (2010) 1639–1648. [22] K. Seo, S. Seo, J.Y. Han, S.H. Ki, S.M. Shin, Toxicol. Appl. Pharmacol. 280 (2014) 314–322. [23] S.H. Jin, J.H. Yang, B.Y. Shin, K. Seo, S.M. Shin, I.J. Cho, S.H. Ki, Toxicol. Appl. Pharmacol. 271 (2013) 95–105. [24] S.H. Hwahng, S.H. Ki, E.J. Bae, H.E. Kim, S.G. Kim, Hepatology 49 (2009) 1913–1925. [25] T. Sanli, K. Linher-Melville, T. Tsakiridis, G. Singh, PLoS One 7 (2012) e32035. [26] Y.-S. Chen, S.-D. Chen, C.-L. Wu, S.-S. Huang, D.-I. Yang, Exp. Neurol. 253 (2014) 63–71. [27] Y. Yang, S. Cuevas, S. Yang, V.A. Villar, C. Escano, L. Asico, P. Yu, X. Jiang, E.J. Weinman, I. Armando, P.A. Jose, Hypertension 64 (2014) 825–832. [28] J.J. Lemasters, A.L. Nieminen, Biosci. Rep. 17 (1997) 281–291. [29] A.V. Gyulkhandanyan, P.S. Pennefather, J. Neurochem. 90 (2004) 405–421. [30] C.J. De Saedeleer, P.E. Porporato, T. Copetti, J. Perez-Escuredo, V.L. Payen, L. Brisson, O. Feron, P. Sonveaux, Oncogene 33 (2014) 4060–4068. [31] Y.N. Kwon, S.M. Shin, I.J. Cho, S.G. Kim, Drug Metab. Dispos. 37 (2009) 1187–1197. [32] V. Petronilli, D. Penzo, L. Scorrano, P. Bernardi, F. Di Lisa, J. Biol. Chem. 276 (2001) 12030–12034. [33] L. Scorrano, D. Penzo, V. Petronilli, F. Pagano, P. Bernardi, J. Biol. Chem. 276 (2001) 12035–12040. [34] J.H. Lee, A.V. Budanov, M. Karin, Cell Metab. 18 (2013) 792–801. [35] M. Ishihara, M. Urushido, K. Hamada, T. Matsumoto, Y. Shimamura, K. Ogata, K. Inoue, Y. Taniguchi, T. Horino, M. Fujieda, S. Fujimoto, Y. Terada, Am. J. Physiol. Lung Cell. Mol. Physiol. 305 (2013) F495–F509. [36] A.A. Eid, D.-Y. Lee, L.J. Roman, K. Khazim, Y. Gorin, Mol. Cell. Biol. 33 (2013) 3439–3460. [37] J.H. Lee, A.V. Budanov, E.J. Park, R. Birse, T.E. Kim, G.A. Perkins, K. Ocorr, M.H. Ellisman, R. Bodmer, E. Bier, M. Karin, Science 327 (2010) 1223–1228. [38] N. Olson, M. Hristova, N.H. Heintz, K.M. Lounsbury, A. van der Vliet, Am. J. Physiol. Lung Cell. Mol. Physiol. 301 (2011) L993–L1002. [39] S.H. Bae, S.H. Sung, S.Y. Oh, J.M. Lim, S.K. Lee, Y.N. Park, H.E. Lee, D. Kang, S.G. Rhee, Cell Metab. 17 (2013) 73–84. [40] S.H. Chang, J. Garcia, J.A. Melendez, M.S. Kilberg, A. Agarwal, Biochem. J. 371 (2003) 877–885. [41] T. Nguyen, P. Nioi, C.B. Pickett, J. Biol. Chem. 284 (2009) 13291–13295. [42] S.K. Niture, R. Khatri, A.K. Jaiswal, Free Radic. Biol. Med. 66 (2014) 36–44.
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003
K. Seo et al. / Cellular Signalling xxx (2015) xxx–xxx [43] K. Itoh, K.I. Tong, M. Yamamoto, Free Radic. Biol. Med. 36 (2004) 1208–1213. [44] A. Nishimoto, N. Kugimiya, T. Hosoyama, T. Enoki, T.S. Li, K. Hamano, Int. J. Oncol. 44 (2014) 2077–2084. [45] S.H. Ro, M. Nam, I. Jang, H.W. Park, H. Park, I.A. Semple, M. Kim, J.S. Kim, H. Park, P. Einat, G. Damari, M. Golikov, E. Feinstein, J.H. Lee, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 7849–7854.
11
[46] D. Kukidome, T. Nishikawa, K. Sonoda, K. Imoto, K. Fujisawa, M. Yano, H. Motoshima, T. Taguchi, T. Matsumura, E. Araki, Diabetes 55 (2006) 120–127. [47] M. Juhaszova, D.B. Zorov, S.-H. Kim, S. Pepe, Q. Fu, K.W. Fishbein, B.D. Ziman, S. Wang, K. Ytrehus, C.L. Antos, E.N. Olson, S.J. Sollott, J. Clin. Invest. 113 (2004) 1535–1549. [48] J. Xi, H. Wang, R.A. Mueller, E.A. Norfleet, Z. Xu, Eur. J. Pharmacol. 604 (2009) 111–116.
Please cite this article as: K. Seo, et al., Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.03.003