Neuroscience Letters 557 (2013) 148–153
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Potential neuroprotective effects of SIRT1 induced by glucose deprivation in PC12 cells Kotaro Fujino, Yurina Ogura, Kazunori Sato, Taku Nedachi ∗ Department of Life Sciences, Graduate School of Life Sciences, Toyo University, Japan
h i g h l i g h t s • SIRT1 induction by glucose deprivation plays an important role for protecting PC12 cells. • Reduced environmental glucose levels affect SIRT1 expression/localization. • The environmental glucose and NGF differentially controlled SIRT1 and FoxO3a.
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Article history: Received 23 August 2013 Received in revised form 9 October 2013 Accepted 20 October 2013 Keywords: Glucose SIRT1 Nerve growth factor FoxO3a PC12
a b s t r a c t Nutrient availability is one of the most important signals regulating cellular fates including cell growth, differentiation, and death. Recent evidence suggests that the NAD+ -dependent histone deacetylase sirtuin 1 (SIRT1) plays a prominent role in linking changes in nutritional availability with cellular fate regulation. SIRT1 expression is observed in neurons, yet the expressional and functional regulation of this protein is not fully understood. In the present study, we examined whether extracellular glucose concentration affects the expression and localization of SIRT1 in PC12 cells. Further, we examined levels of forkhead box O3a (FoxO3a), which is also controlled by changes in extracellular glucose concentration. We observed the total expression levels of SIRT1 and FoxO3a in PC12 cells were reduced when glucose availability increased via gene expressional control, at least in part. Nuclear localization of SIRT1 and FoxO3a was increased by glucose deprivation. Even though the changes in extracellular glucose concentration regulated SIRT1 and FoxO3a in a similar direction, the effects of nerve growth factor on these two proteins were completely different. Finally, we found the potent SIRT1 inhibitor enhanced glucose deprivation-induced cell death. Therefore, we propose that glucose deprivation-induced SIRT1 expression potentially plays a major role in protecting PC12 cells. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Recent evidence indicates that the glucose concentration surrounding cells is crucial for maintaining proper cellular functions. Both excess and deprivation of glucose can be detrimental to cells. Therefore, an optimum glucose concentration is necessary to maintain normal cellular functions. However, how cells monitor “an optimum amount of glucose” remains unclear. Several key intracellular proteins that respond to glucose availability were recently identified. One such protein, the
Abbreviations: BCA, bicinchoninic acid assay; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; FOXO, forkhead transcription factor O; LDH, lactate dehydrogenase; NGF, nerve growth factor; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; RT, room temperature; Sir2, silent information regulator 2; SIRT1, sirtuin 1; Tbs, tris-buffered saline. ∗ Corresponding author at: 1-1-1 Izumino, Oura-gun, Gunma 374-0193, Japan. Tel.: +81 276 82 9028; fax: +81 276 82 9033. E-mail address: [email protected] (T. Nedachi). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.10.050
nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase, silencing information regulator 2 (Sir2), was originally identified in yeast and Caenorhabditis elegans [1,2]. Calorie restriction (or glucose deprivation) promotes Sir2 induction, which resulted in an increased life span for that species. The mammalian homologue of Sir2, sirtuins (SIRT), also responds to calorie restriction, although its impact on longevity is controversial [3–5]. Moreover, recent studies have clearly demonstrated that the substrates of Sir2 or SIRT include histone, as well as other intracellular proteins such as, p53, PGC1a, Hif-1a, Hif-2a, HSF1, and FOXO1-4 [6,7]. More importantly, the Sir2-dependent life span extension observed in C. elegans was dependent on Daf-16, a member of forkhead transcription factor O (FoxO) family [8]. Other important aspects of the FoxO family includes negative regulation by growth factor signaling, especially the phosphatidylinositol 3kinase (PI3K)-PKB/Akt cascade [9]. Akt phosphorylation of the FoxO family is considered the most predominant phenomenon in FoxO inactivation, followed by a change its localization from the nucleus to cytoplasm [9]. Overall, calorie restriction (or glucose deprivation)
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appears to cooperate with growth factor signaling, and thereby controls SIRT1 and FoxO functions. In fact, we previously demonstrated that glucose availability defines the amounts of sirtuin 1 (SIRT1) and FoxO3a in C2C12 skeletal muscle cells [10]. Both these proteins were induced in response to glucose deprivation via gene expressional changes. Furthermore, this mechanism appeared to interact with the insulin-signaling cascade [10]. This type of glucose deprivation was observed in the central nervous system (CNS). For instance, blood supply restriction in brain ischemia resulted in hypoxia and glucose deprivation. The prognosis of ischemia depends, in part, on the duration that the cells are exposed to oxygen and glucose-free conditions and the presence of growth factors that protect cells from death [11,12]. Whether SIRT1 and FOXO are regulated during this process in neuronal cells, in the same way as C2C12 cells, remains elusive. This point of view could be important since it has been reported SIRT1 promotes neuron survival [13,14], whereas FoxO3a promotes neuron apoptosis [15,16]. The rat pheochromocytoma (PC12) cells can be differentiated into neurons with nerve growth factors [17], and are often used for studying neuroprotection [18,19]. Therefore, in the present study, we used PC12 cells to examine glucose-dependent regulation of SIRT1 and FOXO. Moreover, we examined whether the nerve growth factor (NGF) affects these regulating activities. 2. Materials and methods
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IgG conjugated to horseradish peroxidase and an ECL plus detection procedure (GE Healthcare Inc.). Protein concentrations were determined using a bicinchoninic acid assay (BCA, Pierce Biotech. Inc.). 2.4. Immunofluorescence analysis PC12 cells were cultured in the growth medium (DMEM/F-12 supplemented with 10% FBS) for 3 days. The medium was then switched to experimental medium, and the cells were continuously cultured for 72 h. The cells were then fixed with 4% PFA in PBS for 15 min at room temperature (RT). Cells were washed twice with PBS (−) and incubated with 5% normal CS and 0.1% Triton X-100 in PBS (blocking buffer) for 30 min, followed by incubation with primary antibodies (anti-SIRT1; 1:200; Novocastra, Newcastle, UK; anti-FoxO3 antibody; 1:250; Cell Signaling Technology) for 2 h at RT. After washing the cells thrice with PBS, Alexa Fluor-conjugated secondary antibodies (Alexa Fluor 488 or 594; 1:500 dilution; Invitrogen Corp.) and 5 g/ml Hoechst 33258 in blocking buffer were added, and the cells were incubated for 1 h at RT. Again, cells were washed thrice with PBS and then observed under LSM five Pascal/Axiovert 200 confocal microscopes (Carl Zeiss, Oberkochen, Germany). The images were analyzed by using fluorescence area intensity measurements in the nucleus and in cytoplasm for SIRT1 or FoxO3a. The average of nuclear/cytoplasmic ratios +/− SEM for 10–35 cells was shown in the graph.
2.1. Materials 2.5. Glucose measurement The western blot detection kit (ECL plus or ECL prime detection reagents) was purchased from GE Healthcare Inc. (Rockford, IL, USA). Dulbecco’s Modified Eagle Medium (DMEM), penicillin/streptomycin, and trypsin-EDTA were purchased from Nakaraitesque (Kyoto, Japan). Cell culture equipment was obtained from BD Biosciences (San Jose, CA, USA). Calf Serum (CS) and Fetal Bovine Serum (FBS) were obtained from BioWest (Nuaille, France). Immobilon-P was obtained from Millipore Corp. (Bedford, MA, USA). Unless otherwise noted, all chemicals were of the purest grade available from Nakaraitesque, Sigma Chemicals (St. Louis, MO, USA) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 2.2. Cell culture PC12 cells were obtained through a generous gift from Dr. ShinIchiro Takahashi (The University of Tokyo, Tokyo, Japan). The cells were maintained in DMEM supplemented with 10% FBS, 30 g/ml penicillin, and 100 g/ml streptomycin (growth medium) at 37 ◦ C under a 5% CO2 atmosphere. For biochemical studies, cells were grown on 6-well plates (Orange Scientific, Braine-l’Alleud, Belgium) at a density of 5 × 104 cells/well in 3 ml of growth medium, or on 96-well plates (Orange Scientific) at a density of 5 × 103 cells/well in 0.2 ml of growth medium. Three days after plating, cells typically reached 50–70% confluence (Day 0). Differentiation was then induced by switching the growth medium to DMEM containing either 5 mM glucose (LG-DMEM) or 22.5 mM glucose (HG-DMEM) supplemented with 10–100 ng/ml NGF, 30 g/ml penicillin, and 100 g/ml streptomycin.
Glucose concentration in the cultured media were measured using a determiner GLE kit (Kyowa Medex, Tokyo, Japan). 2.6. Real time PCR Fluorescence real time PCR analysis was performed using StepOne instrument (Life Technologies Corporation, Grand Island, NY, USA) and SYBR Green detection kit according to the manufacture’s procedure (Life Technologies or KAPA Biosystems Inc., Woburn, MA, USA). PCR primers for measuring each gene included the following: SIRT1, 5 -TTT CAG AAC CAC CAA AGC G-3 and 5 TCC CAC AGG AAA CAG AAA CC-3 ; FoxO3a, 5 -TGC TAA GCA GGC CTC ATC TCA A-3 and 5 -AGA TGG CGT GGG AGT CAC AA-3 ; and GAPDH, 5 -GGC ACA GTC AAG GCT GAG AAT G-3 and 5 -ATG GTG GTG AAG ACG CCA GTA-3 . 2.6.1. Measurement of cell death PC12 cells were seeded on 96-well plates and differentiated as described previously. The percentage of cell death was evaluated using the lactose dehydrogenase (LDH) plus kit (Roche Diagnostics K.K., Basel, Switzerland) according to the manufacturer’s protocol. 2.7. Statistical analysis Comparison among treatment groups was tested using one-way ANOVA and a post hoc Tukey test or Student’s t-test. Differences in which p < 0.05 were considered statistically significant.
2.3. Western blotting
3. Results
The expression and phosphorylation of each protein were analyzed by western blot analysis using previously described methods [10]. Detection of each protein was achieved with 1 h incubation with a 1:1000 dilution of primary antibody (anti-SIRT1, anti-FoxO3a, anti-cleaved caspase-3; Cell Signaling Technology, Danvers, MA, USA). Specific total proteins were visualized after subsequent incubation with a 1:5000 dilution of anti-mouse or rabbit
We previously reported the extracellular glucose levels define SIRT1 and FoxO3a levels in C2C12 skeletal muscle cells [10]. Therefore, we initially examined whether glucose deprivation affects SIRT1 and FoxO3 expression levels in PC12 cells. Before executing the entire experiment, we evaluated glucose consumption. In our experimental condition (see Section 2), cells were either grown in LG-DMEM or HG-DMEM; glucose concentration in these media
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Fig. 1. Effects of glucose and NGF on SIRT1 expression and localization in PC12 cells. (A–E) PC12 cells were differentiated under either low (LG) or high (HG) glucose conditions in the presence of the indicated amounts of NGF for 72 h. (A) Cell lysates were prepared, and the same amounts of protein samples were subjected to western blotting using anti-SIRT1 antibody. (B) Densitometric analysis of (A) (*p < 0.05, **p < 0.01, n = 3, one-way ANOVA). (C) Total RNA were prepared and subjected to real time PCR analysis for evaluating SIRT1 gene expression (*p < 0.05, **p < 0.01, n = 3, one-way ANOVA). (D) Differentiated PC12 cells under either LG or HG conditions were fixed and immunostained using anti-SIRT1 antibody (panel c, d, i, j). Hoechst 33342 staining was performed at the same time to confirm the position of the nucleus (panel a, b, g, h). Merged images are shown in panel e, f, k, l. All experiments were performed at least thrice, and similar results were obtained. (E) Signal intensity of nucleic SIRT1 and cytosolic SIRT1 in each panel were measured, and nuclear/cytoplasmic ratios were shown (**p < 0.01, n = 10–12, unpaired t-test).
was gradually decreased at an approximate rate of 0.4 g l−1 and 24 h−1 , which indicated LG-DMEM was completely deprived of glucose after 72 h of culture (Supplementary Fig. 1A). For elucidating the interaction between glucose deprivation and NGF, we initially attempted to compare between 0 ng/ml and 100 ng/ml NGF; however, it was difficult to maintain cells for 24 h without serum or NGF (data not shown). Thus, we decided to use 10 ng/ml NGF (presumably less effective concentration compared to 100 ng/ml NGF) instead of 0 ng/ml NGF. To confirm if 10 ng/ml NGF was suitable for following experiments, we evaluated the effects of different concentration of NGF on cell viability and differentiation. PC12 cell death was indeed attenuated in the presence of 10 ng/ml of NGF, as confirmed by the LDH assay, although the cell death was significantly higher in LG conditions than in HG conditions (Supplementary Fig. 1B). NGF treatment at 100 ng/ml showed more prominent effects on cell differentiation than treatment at 10 ng/ml, as confirmed by MAP2 expression analysis (Supplementary Fig. 1C) and measurement of neurite outgrowth (Supplementary Fig. 1D and E). It should be noted that the neurite outgrowth in LG condition was significantly increased compared to that in HG condition (Supplementary Fig. 1E). Thus, to elucidate the interaction between glucose deprivation and NGF, both 10 ng/ml NGF and 100 ng/ml NGF were used in the following experiments. To examine the effects of glucose deprivation and NGF on SIRT1 expression, PC12 cells were cultured under the condition of either
LG-DMEM or HG-DMEM for 24–72 h. Expression levels of each protein were subsequently evaluated by western blotting analysis using specific antibodies. When cells were maintained in LGDMEM with 10 ng/ml NGF for 72 h, SIRT1 levels were significantly increased compared to when cells were maintained in HG-DMEM with 10 ng/ml NGF (Fig. 1A and B). The effect of NGF on this change was observed only in glucose-deprived conditions; 100 ng/ml NGF treatment significantly increased SIRT1 levels (Fig. 1A and B). On the other hand, 24–48 h of maintenance with LG-DMEM had no significant effect on SIRT1 levels either with or without high concentrations of NGF (data not shown). Real time PCR analysis showed that these changes in SIRT1 protein levels were correlated with SIRT1 gene expression (Fig. 1C). Furthermore, we analyzed whether intracellular localization of SIRT1 was affected in cells exposed to LG-DMEM or HG-DMEM for 72 h. In HG-DMEM conditions, the predominant SIRT1 localization was observed in cytosolic fractions, whereas in LG-DMEM conditions, most of the SIRT1 signal was observed in the nucleus (Fig. 1D and E). In addition, the nuclear localization of SIRT1 was significantly reduced by increasing NGF concentration from 10 ng/ml to 100 ng/ml (Fig. 1E). These experiments suggested that PC12 cells possessed glucose responsiveness, and glucose availability influenced both the level and localization of SIRT1. As we previously reported, regulation of FoxO3a and SIRT1 levels in C2C12 myotubes was similarly controlled by changes in
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Fig. 2. Effects of glucose and NGF on FoxO3a expression and localization in PC12 cells. (A–E) PC12 cells were differentiated under either LG or HG conditions in the presence of indicated amounts of NGF for 72 h. (A) Cell lysates were prepared as described in Section 2, and the same amounts of protein samples were subjected to western blotting using anti-FoxO3a antibody. (B) Densitometric analysis of (A) (*p < 0.05, n = 3, one-way ANOVA). (C) Total RNA were prepared and subjected to real time PCR analysis for evaluating FoxO3a gene expression (*p < 0.05, n = 3). (D) Differentiated PC12 cells under either LG or HG conditions were fixed and immunostained using anti-FoxO3a antibody (panel c, d, i, j). Hoechst 33342 staining was performed at the same time to confirm the position of the nucleus (panel a, b, g, h). Merged images were shown in panel e, f, k, l. All experiments were performed at least thrice, and similar results were obtained. (E) Signal intensity of nucleic FoxO3a and cytosolic FoxO3a in each panel were measured, and nuclear/cytoplasmic ratios were shown (**p < 0.01, n = 30–35, unpaired t-test).
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100 ng/ml NGF reversed this effect, suggesting that NGF prevents LG-induced FoxO3a accumulation in the nucleus (Fig. 2D and E). Finally, we examined the role of glucose deprivation-dependent SIRT1 induction in PC12 cells. Potent SIRT1 inhibitors, sirtinol or Ex527, were administered to PC12 cells cultured in either LG or HG conditions in the presence of 10 ng/ml NGF. Cell death was then evaluated by LDH assay. We observed that sirtinol or Ex527 had no apparent effect on cell death in HG conditions, but it significantly enhanced cell death in LG conditions (Fig. 3A and B). Cleaved caspase-3, which is often used as an index of apoptosis, was induced by glucose deprivation (Fig. 3C). This glucose deprivationinduced apoptosis, as assessed by measuring cleaved caspase-3 levels, was further potentiated by sirtinol treatment (Fig. 3C). These results suggested that even though glucose deprivation enhanced cell death, this condition also induced SIRT1 expression to protect cells from death (Fig. 3D).
4. Discussion
Fig. 3. Effects of SIRT1 inhibitor on PC12 cell death. (A–C) PC12 cells were differentiated under either LG conditions or HG conditions in the presence of 10 ng/ml NGF for 72 h. Indicated amounts of sirtinol (A and C) or Ex527 (B) were added to cell at 48 h. (A and B) Total cell death was measured by LDH assay (*p < 0.05, n = 3, oneway ANOVA). (C) The cell lysates were prepared, and apoptosis of PC12 cells was evaluated by measuring cleaved caspase-3 levels (*p < 0.05, n = 4, one-way ANOVA). (D) Schematic depiction of the present study. Reduced environmental glucose levels had biphasic effects: induction of cell death that was perhaps independent of FoxO3 regulation and reduction of cell death via SIRT1 induction.
glucose availability [10]. Therefore, we examined whether FOXO level in PC12 cells were affected by extracellular glucose deprivation. Similar to the SIRT1 results, FoxO3a levels were significantly increased in HG-DMEM with 10 ng/ml (Fig. 2A and B), which was accompanied by increased gene expression, as assessed by real time PCR analysis (Fig. 2C). Remarkably, the effects of NGF were unlike those for SIRT1, the addition of 100 ng/ml NGF in LG-DMEM significantly decreased FoxO3a level compared to the addition of 10 ng/ml NGF. (Fig. 2A and B). However, gene expression of FoxO3a was not influenced by NGF treatment (Fig. 2A and B). These results clearly indicated that increasing both glucose availability and NGF decreased FoxO3a expression, but the underlying mechanisms were completely different. Immunofluorescence studies revealed that glucose deprivation changed FoxO3a localization from the cytosol to the nucleus (Fig. 2D and E). Treatment with
Reduced glucose levels surrounding neurons can change the direction of their cellular fate towards death. This is an especially critical issue during ischemia. Our present findings strongly suggest that two glucose-sensitive proteins, SIRT1 and FoxO3a, are affected by reduced environmental glucose levels, which enhance their activities. This occurs even though the two proteins have opposite biological effects; SIRT1 promotes neuron survival [13,14], whereas FoxO3a promotes neuron apoptosis [15,16]. Intriguingly, NGF did not affect SIRT1 protein levels, but decreased FoxO3a protein levels, even though the impact of FoxO3a repression on cell death was minimal in PC12 cells. Overall, our results provide new insights in that glucose deprivation not only promotes cell death, but also exerts neuroprotective mechanisms by inducing SIRT1. Glucose deprivation is one of the major consequences of ischemia that subsequently induces neuronal cell death. The oxygen-glucose deprivation (OGD) model is widely used to investigate ischemia pathogenesis [20]. Certainly, the OGD model has an advantage for modeling ischemia, but distinguishing oxygen and glucose deprivation is necessary for understanding the detailed mechanisms of pathogenesis for these diseases. Besides, several reports have suggested abnormalities in brain glucose utilization in Alzheimer’s disease and amyotrophic lateral sclerosis [21–23]. Thus, our glucose deprivation model provides an important viewpoint on the pathogenesis of neuronal disorders. Liu Y et al. showed that glucose deprivation induced mitochondrial dysfunction and accumulation of reactive oxygen species (ROS), which promoted both necrosis and apoptosis in PC12 cells [24]. In this study, we confirmed that 48 h incubation of PC12 cells with LG-DMEM diminished glucose concentration in the medium to approximately zero, and initiated cell death, despite the presence of NGF (Supplementary Fig. 1A). Thus, glucose deprivation apparently increased PC12 cell death. Recent findings regarding SIRT1 have revealed that SIRT1 protein levels vary dramatically with nutrient availability in various tissues and cell lines [10,25,26]. On the other hand, whether this change is mediated by transcriptional control is still controversial [27,28]. In the present study, we observed SIRT1 gene expressional changes in response to glucose deprivation, which indicated changes that in glucose availability indeed regulates transcriptional properties for SIRT1 gene expression, at least in PC12 cells (Fig. 1C). In addition, we found that NGF treatment induced SIRT1 gene and protein expression, only in the absence of glucose (Fig. 1A and B). This finding is consistent with our previous observations that demonstrated interactions between glucose deprivation and insulin in C2C12 myocytes [10]. Overall, these interactions between extracellular glucose levels and growth factors
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affecting SIRT1 appear to be conserved among different types of cells. Interestingly, Sundaresan et al. [29] reported that SIRT1 deacetylates and activates Akt and PDK1, both of which are important molecules in growth factor signaling. This suggested that glucose deprivation- and growth factor-dependent SIRT1 induction involves a positive feedback mechanism to enhance growth factor signaling. Glucose deprivation also changed the intracellular localization of SIRT1—from cytosol to nucleus (Fig. 1D and E). The impact of this change in SIRT1 localization in PC12 cells is currently unknown; however, Jin et al. proposed that cytoplasm-localized SIRT1 enhances apoptosis [30], thus, nuclear-localized SIRT1 may function to prevent apoptosis in PC12 cells. It is well documented that induction of FoxO3a leads to ROS overproduction and stimulates apoptosis [31,32]. Moreover, it has also been established nucleic FoxO3a accumulation is directly involved in apoptosis [33,34]. However, the impact of reduction and locational changes of FoxO3a on cell viability appeared to be minimal, at least in PC12 cells because the cell death ratio was not significantly different between 10 ng/ml and 100 ng/ml NGF treatments. The biological roles of FoxO3a expression changes in PC12 cells are now under investigation. We found that inhibition of SIRT1 in the LG condition significantly enhanced cell death in PC12 cells. This result might not be surprising considering that resveratrol can inhibit betaamyloid–induced cell apoptosis through SIRT1 upregulation in PC12 cells [35]. However, our present results suggested that reduced environmental glucose levels had biphasic effects. Induction of cell death that was perhaps independent of FoxO3a regulation and the reduction of cell death via SIRT1 induction and nuclear translocation (Fig. 3D). The disturbance of this balance may directly determine neuronal cell fates. Acknowledgements We are deeply grateful to Dr. Shin-Ichiro Takahashi for PC12 cells. We also appreciate Dr. Masugi Nishihara for many constructive comments. This work was supported by Grants-in-Aid for Scientific Research (S) 23228004 and (C) 24580147 from the Japan Society for the Promotion of Science. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.neulet.2013.10.050. References [1] J. Rine, I. Herskowitz, Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae, Genetics 116 (1987) 9–22. [2] H.A. Tissenbaum, L. Guarente, Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans, Nature 410 (2001) 227–230. [3] L. Bordone, D. Cohen, A. Robinson, M.C. Motta, E. van Veen, A. Czopik, A.D. Steele, H. Crowe, S. Marmor, J. Luo, W. Gu, L. Guarente, SIRT1 transgenic mice show phenotypes resembling calorie restriction, Aging Cell 6 (2007) 759–767. [4] T. Finkel, C.X. Deng, R. Mostoslavsky, Recent progress in the biology and physiology of sirtuins, Nature 460 (2009) 587–591. [5] D. Herranz, M. Serrano, SIRT1: recent lessons from mouse models, Nat. Rev. Cancer 10 (2010) 819–823. [6] M.C. Haigis, D.A. Sinclair, Mammalian sirtuins: biological insights and disease relevance, Ann. Rev. Pathol. 5 (2010) 253–295. [7] M. Jiang, J. Wang, J. Fu, L. Du, H. Jeong, T. West, L. Xiang, Q. Peng, Z. Hou, H. Cai, T. Seredenina, N. Arbez, S. Zhu, K. Sommers, J. Qian, J. Zhang, S. Mori, X.W. Yang, K.L. Tamashiro, S. Aja, T.H. Moran, R. Luthi-Carter, B. Martin, S. Maudsley, M.P. Mattson, R.H. Cichewicz, C.A. Ross, D.M. Holtzman, D. Krainc, W. Duan, Neuroprotective role of Sirt1 in mammalian models of Huntington’s disease through activation of multiple Sirt1 targets, Nat. Med. 18 (2011) 153–158.
153
[8] M.E. Giannakou, L. Partridge, The interaction between FOXO and SIRT1: tipping the balance towards survival, Trends Cell Biol. 14 (2004) 408–412. [9] B.M. Burgering, G.J. Kops, Cell cycle and death control: long live Forkheads, Trends Biochem. Sci. 27 (2002) 352–360. [10] T. Nedachi, A. Kadotani, M. Ariga, H. Katagiri, M. Kanzaki, Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes, Am. J. Physiol. Endocrinol. Metab. 294 (2008) E668–E678. [11] A. Schurr, Energy metabolism, stress hormones and neural recovery from cerebral ischemia/hypoxia, Neurochem. Int. 41 (2002) 1–8. [12] B.C. White, J.M. Sullivan, D.J. DeGracia, B.J. O’Neil, R.W. Neumar, L.I. Grossman, J.A. Rafols, G.S. Krause, Brain ischemia and reperfusion: molecular mechanisms of neuronal injury, J. Neurol. Sci. 179 (2000) 1–33. [13] T. Araki, Y. Sasaki, J. Milbrandt, Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration, Science 305 (2004) 1010–1013. [14] B.L. Tang, SIRT1, neuronal cell survival and the insulin/IGF-1 aging paradox, Neurobiol. Aging 27 (2006) 501–505. [15] J. Gilley, P.J. Coffer, J. Ham, FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons, J. Cell Biol. 162 (2003) 613–622. [16] C. Barthélémy, C.E. Henderson, B. Pettmann, Foxo3a induces motoneuron death through the Fas pathway in cooperation with JNK, BMC Neurosci. 5 (2004) 48. [17] P.W. Gunning, G.E. Landreth, P. Layer, M. Ignatius, E.M. Shooter, Nerve growth factor-induced differentiation of PC12 cells: evaluation of changes in RNA and DNA metabolism, J. Neurosci. 1 (1981) 368–379. [18] R. Tabakman, P. Lazarovici, R. Kohen, Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia, J. Neurosci. Res. 68 (2002) 463–469. [19] G. Li, R. Ma, C. Huang, Q. Tang, Q. Fu, H. Liu, B. Hu, J. Xiang, Protective effect of erythropoietin on beta-amyloid-induced PC12 cell death through antioxidant mechanisms, Neurosci. Lett. 442 (2008) 143–147. [20] R. Tabakman, H. Jiang, I. Shahar, H. Arien-Zakay, R.A. Levine, P. Lazarovici, Neuroprotection by NGF in the PC12 in vitro OGD model: involvement of mitogen-activated protein kinases and gene expression, Ann. N.Y. Acad. Sci. 1053 (2005) 84–96. [21] S. Hoyer, Brain oxidative energy and related metabolism, neuronal stress, and Alzheimer’s disease: a speculative synthesis, J. Geriatr. Psychiatry Neurol. 6 (1993) 3–13. [22] R.J. Mark, Z. Pang, J.W. Geddes, K. Uchida, M.P. Mattson, Amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation, J. Neurosci. 17 (1997) 1046–1054. [23] M.C. Dalakas, J. Hatazawa, R.A. Brooks, G. Di Chiro, Lowered cerebral glucose utilization in amyotrophic lateral sclerosis, Ann. Neurol. 22 (1987) 580–586. [24] Y. Liu, X.D. Song, W. Liu, T.Y. Zhang, J. Zuo, Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line, J. Cell Mol. Med. 7 (2003) 49–53. [25] H.Y. Cohen, C. Miller, K.J. Bitterman, N.R. Wall, B. Hekking, B. Kessler, K.T. Howitz, M. Gorospe, R. de Cabo, D.A. Sinclair, Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase, Science 305 (2004) 390–392. [26] S. Kume, T. Uzu, K. Horiike, M. Chin-Kanasaki, K. Isshiki, S. Araki, T. Sugimoto, M. Haneda, A. Kashiwagi, D. Koya, Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney, J. Clin. Invest. 120 (2010) 1043–1055. [27] S. Nemoto, M.M. Fergusson, T. Finkel, Nutrient availability regulates SIRT1 through a forkhead-dependent pathway, Science 306 (2004) 2105–2108. [28] Y. Kanfi, V. Peshti, Y.M. Gozlan, M. Rathaus, R. Gil, H.Y. Cohen, Regulation of SIRT1 protein levels by nutrient availability, FEBS Lett. 582 (2008) 2417–2423. [29] N.R. Sundaresan, V.B. Pillai, D. Wolfgeher, S. Samant, P. Vasudevan, V. Parekh, H. Raghuraman, J.M. Cunningham, M. Gupta, M.P. Gupta, The deacetylase SIRT1 promotes membrane localization and activation of Akt and PDK1 during tumorigenesis and cardiac hypertrophy, Sci. Signal. 4 (2011) ra46. [30] Q. Jin, T. Yan, X. Ge, C. Sun, X. Shi, Q. Zhai, Cytoplasm-localized SIRT1 enhances apoptosis, J. Cell Physiol. 213 (2007) 88–97. [31] P. Obexer, K. Geiger, P.F. Ambros, B. Meister, M.J. Ausserlechner, FKHRL1mediated expression of Noxa and Bim induces apoptosis via the mitochondria in neuroblastoma cells, Cell Death Differ. 14 (2007) 534–547. [32] P. Obexer, J. Hagenbuchner, T. Unterkircher, N. Sachsenmaier, C. Seifarth, G. Böck, V. Porto, K. Geiger, M. Ausserlechner, Repression of BIRC5/survivin by FOXO3/FKHRL1 sensitizes human neuroblastoma cells to DNA damage-induced apoptosis, Mol. Biol. Cell 20 (2009) 2041–2048. [33] W.H. Zheng, S. Kar, R. Quirion, Insulin-like growth factor-1-induced phosphorylation of the forkhead family transcription factor FKHRL1 is mediated by Akt kinase in PC12 cells, J. Biol. Chem. 275 (2000) 39152–39158. [34] J. Chintapalli, S. Yang, D. Opawumi, S.R. Goyal, N. Shamsuddin, A. Malhotra, K. Reiss, L.G. Meggs, Inhibition of wild-type p66ShcA in mesangial cells prevents glycooxidant-dependent FOXO3a regulation and promotes the survival phenotype, Am. J. Physiol. Renal Physiol. 292 (2007) F523–F530. [35] X. Feng, N. Liang, D. Zhu, Q. Gao, L. Peng, H. Dong, Q. Yue, H. Liu, L. Bao, J. Zhang, J. Hao, Y. Gao, X. Yu, J. Sun, Resveratrol inhibits -amyloid-induced neuronal apoptosis through regulation of SIRT1-ROCK1 signaling pathway, PLoS One 8 (2013) e59888.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Potential neuroprotective effects of SIRT1 induced by glucose deprivation in PC12 cells Kotaro Fujino, Yurina Ogura, Kazunori Sato, Taku Nedachi ∗ Department of Life Sciences, Graduate School of Life Sciences, Toyo University, Japan
h i g h l i g h t s • SIRT1 induction by glucose deprivation plays an important role for protecting PC12 cells. • Reduced environmental glucose levels affect SIRT1 expression/localization. • The environmental glucose and NGF differentially controlled SIRT1 and FoxO3a.
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Article history: Received 23 August 2013 Received in revised form 9 October 2013 Accepted 20 October 2013 Keywords: Glucose SIRT1 Nerve growth factor FoxO3a PC12
a b s t r a c t Nutrient availability is one of the most important signals regulating cellular fates including cell growth, differentiation, and death. Recent evidence suggests that the NAD+ -dependent histone deacetylase sirtuin 1 (SIRT1) plays a prominent role in linking changes in nutritional availability with cellular fate regulation. SIRT1 expression is observed in neurons, yet the expressional and functional regulation of this protein is not fully understood. In the present study, we examined whether extracellular glucose concentration affects the expression and localization of SIRT1 in PC12 cells. Further, we examined levels of forkhead box O3a (FoxO3a), which is also controlled by changes in extracellular glucose concentration. We observed the total expression levels of SIRT1 and FoxO3a in PC12 cells were reduced when glucose availability increased via gene expressional control, at least in part. Nuclear localization of SIRT1 and FoxO3a was increased by glucose deprivation. Even though the changes in extracellular glucose concentration regulated SIRT1 and FoxO3a in a similar direction, the effects of nerve growth factor on these two proteins were completely different. Finally, we found the potent SIRT1 inhibitor enhanced glucose deprivation-induced cell death. Therefore, we propose that glucose deprivation-induced SIRT1 expression potentially plays a major role in protecting PC12 cells. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Recent evidence indicates that the glucose concentration surrounding cells is crucial for maintaining proper cellular functions. Both excess and deprivation of glucose can be detrimental to cells. Therefore, an optimum glucose concentration is necessary to maintain normal cellular functions. However, how cells monitor “an optimum amount of glucose” remains unclear. Several key intracellular proteins that respond to glucose availability were recently identified. One such protein, the
Abbreviations: BCA, bicinchoninic acid assay; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; FOXO, forkhead transcription factor O; LDH, lactate dehydrogenase; NGF, nerve growth factor; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; RT, room temperature; Sir2, silent information regulator 2; SIRT1, sirtuin 1; Tbs, tris-buffered saline. ∗ Corresponding author at: 1-1-1 Izumino, Oura-gun, Gunma 374-0193, Japan. Tel.: +81 276 82 9028; fax: +81 276 82 9033. E-mail address: [email protected] (T. Nedachi). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.10.050
nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase, silencing information regulator 2 (Sir2), was originally identified in yeast and Caenorhabditis elegans [1,2]. Calorie restriction (or glucose deprivation) promotes Sir2 induction, which resulted in an increased life span for that species. The mammalian homologue of Sir2, sirtuins (SIRT), also responds to calorie restriction, although its impact on longevity is controversial [3–5]. Moreover, recent studies have clearly demonstrated that the substrates of Sir2 or SIRT include histone, as well as other intracellular proteins such as, p53, PGC1a, Hif-1a, Hif-2a, HSF1, and FOXO1-4 [6,7]. More importantly, the Sir2-dependent life span extension observed in C. elegans was dependent on Daf-16, a member of forkhead transcription factor O (FoxO) family [8]. Other important aspects of the FoxO family includes negative regulation by growth factor signaling, especially the phosphatidylinositol 3kinase (PI3K)-PKB/Akt cascade [9]. Akt phosphorylation of the FoxO family is considered the most predominant phenomenon in FoxO inactivation, followed by a change its localization from the nucleus to cytoplasm [9]. Overall, calorie restriction (or glucose deprivation)
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appears to cooperate with growth factor signaling, and thereby controls SIRT1 and FoxO functions. In fact, we previously demonstrated that glucose availability defines the amounts of sirtuin 1 (SIRT1) and FoxO3a in C2C12 skeletal muscle cells [10]. Both these proteins were induced in response to glucose deprivation via gene expressional changes. Furthermore, this mechanism appeared to interact with the insulin-signaling cascade [10]. This type of glucose deprivation was observed in the central nervous system (CNS). For instance, blood supply restriction in brain ischemia resulted in hypoxia and glucose deprivation. The prognosis of ischemia depends, in part, on the duration that the cells are exposed to oxygen and glucose-free conditions and the presence of growth factors that protect cells from death [11,12]. Whether SIRT1 and FOXO are regulated during this process in neuronal cells, in the same way as C2C12 cells, remains elusive. This point of view could be important since it has been reported SIRT1 promotes neuron survival [13,14], whereas FoxO3a promotes neuron apoptosis [15,16]. The rat pheochromocytoma (PC12) cells can be differentiated into neurons with nerve growth factors [17], and are often used for studying neuroprotection [18,19]. Therefore, in the present study, we used PC12 cells to examine glucose-dependent regulation of SIRT1 and FOXO. Moreover, we examined whether the nerve growth factor (NGF) affects these regulating activities. 2. Materials and methods
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IgG conjugated to horseradish peroxidase and an ECL plus detection procedure (GE Healthcare Inc.). Protein concentrations were determined using a bicinchoninic acid assay (BCA, Pierce Biotech. Inc.). 2.4. Immunofluorescence analysis PC12 cells were cultured in the growth medium (DMEM/F-12 supplemented with 10% FBS) for 3 days. The medium was then switched to experimental medium, and the cells were continuously cultured for 72 h. The cells were then fixed with 4% PFA in PBS for 15 min at room temperature (RT). Cells were washed twice with PBS (−) and incubated with 5% normal CS and 0.1% Triton X-100 in PBS (blocking buffer) for 30 min, followed by incubation with primary antibodies (anti-SIRT1; 1:200; Novocastra, Newcastle, UK; anti-FoxO3 antibody; 1:250; Cell Signaling Technology) for 2 h at RT. After washing the cells thrice with PBS, Alexa Fluor-conjugated secondary antibodies (Alexa Fluor 488 or 594; 1:500 dilution; Invitrogen Corp.) and 5 g/ml Hoechst 33258 in blocking buffer were added, and the cells were incubated for 1 h at RT. Again, cells were washed thrice with PBS and then observed under LSM five Pascal/Axiovert 200 confocal microscopes (Carl Zeiss, Oberkochen, Germany). The images were analyzed by using fluorescence area intensity measurements in the nucleus and in cytoplasm for SIRT1 or FoxO3a. The average of nuclear/cytoplasmic ratios +/− SEM for 10–35 cells was shown in the graph.
2.1. Materials 2.5. Glucose measurement The western blot detection kit (ECL plus or ECL prime detection reagents) was purchased from GE Healthcare Inc. (Rockford, IL, USA). Dulbecco’s Modified Eagle Medium (DMEM), penicillin/streptomycin, and trypsin-EDTA were purchased from Nakaraitesque (Kyoto, Japan). Cell culture equipment was obtained from BD Biosciences (San Jose, CA, USA). Calf Serum (CS) and Fetal Bovine Serum (FBS) were obtained from BioWest (Nuaille, France). Immobilon-P was obtained from Millipore Corp. (Bedford, MA, USA). Unless otherwise noted, all chemicals were of the purest grade available from Nakaraitesque, Sigma Chemicals (St. Louis, MO, USA) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 2.2. Cell culture PC12 cells were obtained through a generous gift from Dr. ShinIchiro Takahashi (The University of Tokyo, Tokyo, Japan). The cells were maintained in DMEM supplemented with 10% FBS, 30 g/ml penicillin, and 100 g/ml streptomycin (growth medium) at 37 ◦ C under a 5% CO2 atmosphere. For biochemical studies, cells were grown on 6-well plates (Orange Scientific, Braine-l’Alleud, Belgium) at a density of 5 × 104 cells/well in 3 ml of growth medium, or on 96-well plates (Orange Scientific) at a density of 5 × 103 cells/well in 0.2 ml of growth medium. Three days after plating, cells typically reached 50–70% confluence (Day 0). Differentiation was then induced by switching the growth medium to DMEM containing either 5 mM glucose (LG-DMEM) or 22.5 mM glucose (HG-DMEM) supplemented with 10–100 ng/ml NGF, 30 g/ml penicillin, and 100 g/ml streptomycin.
Glucose concentration in the cultured media were measured using a determiner GLE kit (Kyowa Medex, Tokyo, Japan). 2.6. Real time PCR Fluorescence real time PCR analysis was performed using StepOne instrument (Life Technologies Corporation, Grand Island, NY, USA) and SYBR Green detection kit according to the manufacture’s procedure (Life Technologies or KAPA Biosystems Inc., Woburn, MA, USA). PCR primers for measuring each gene included the following: SIRT1, 5 -TTT CAG AAC CAC CAA AGC G-3 and 5 TCC CAC AGG AAA CAG AAA CC-3 ; FoxO3a, 5 -TGC TAA GCA GGC CTC ATC TCA A-3 and 5 -AGA TGG CGT GGG AGT CAC AA-3 ; and GAPDH, 5 -GGC ACA GTC AAG GCT GAG AAT G-3 and 5 -ATG GTG GTG AAG ACG CCA GTA-3 . 2.6.1. Measurement of cell death PC12 cells were seeded on 96-well plates and differentiated as described previously. The percentage of cell death was evaluated using the lactose dehydrogenase (LDH) plus kit (Roche Diagnostics K.K., Basel, Switzerland) according to the manufacturer’s protocol. 2.7. Statistical analysis Comparison among treatment groups was tested using one-way ANOVA and a post hoc Tukey test or Student’s t-test. Differences in which p < 0.05 were considered statistically significant.
2.3. Western blotting
3. Results
The expression and phosphorylation of each protein were analyzed by western blot analysis using previously described methods [10]. Detection of each protein was achieved with 1 h incubation with a 1:1000 dilution of primary antibody (anti-SIRT1, anti-FoxO3a, anti-cleaved caspase-3; Cell Signaling Technology, Danvers, MA, USA). Specific total proteins were visualized after subsequent incubation with a 1:5000 dilution of anti-mouse or rabbit
We previously reported the extracellular glucose levels define SIRT1 and FoxO3a levels in C2C12 skeletal muscle cells [10]. Therefore, we initially examined whether glucose deprivation affects SIRT1 and FoxO3 expression levels in PC12 cells. Before executing the entire experiment, we evaluated glucose consumption. In our experimental condition (see Section 2), cells were either grown in LG-DMEM or HG-DMEM; glucose concentration in these media
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Fig. 1. Effects of glucose and NGF on SIRT1 expression and localization in PC12 cells. (A–E) PC12 cells were differentiated under either low (LG) or high (HG) glucose conditions in the presence of the indicated amounts of NGF for 72 h. (A) Cell lysates were prepared, and the same amounts of protein samples were subjected to western blotting using anti-SIRT1 antibody. (B) Densitometric analysis of (A) (*p < 0.05, **p < 0.01, n = 3, one-way ANOVA). (C) Total RNA were prepared and subjected to real time PCR analysis for evaluating SIRT1 gene expression (*p < 0.05, **p < 0.01, n = 3, one-way ANOVA). (D) Differentiated PC12 cells under either LG or HG conditions were fixed and immunostained using anti-SIRT1 antibody (panel c, d, i, j). Hoechst 33342 staining was performed at the same time to confirm the position of the nucleus (panel a, b, g, h). Merged images are shown in panel e, f, k, l. All experiments were performed at least thrice, and similar results were obtained. (E) Signal intensity of nucleic SIRT1 and cytosolic SIRT1 in each panel were measured, and nuclear/cytoplasmic ratios were shown (**p < 0.01, n = 10–12, unpaired t-test).
was gradually decreased at an approximate rate of 0.4 g l−1 and 24 h−1 , which indicated LG-DMEM was completely deprived of glucose after 72 h of culture (Supplementary Fig. 1A). For elucidating the interaction between glucose deprivation and NGF, we initially attempted to compare between 0 ng/ml and 100 ng/ml NGF; however, it was difficult to maintain cells for 24 h without serum or NGF (data not shown). Thus, we decided to use 10 ng/ml NGF (presumably less effective concentration compared to 100 ng/ml NGF) instead of 0 ng/ml NGF. To confirm if 10 ng/ml NGF was suitable for following experiments, we evaluated the effects of different concentration of NGF on cell viability and differentiation. PC12 cell death was indeed attenuated in the presence of 10 ng/ml of NGF, as confirmed by the LDH assay, although the cell death was significantly higher in LG conditions than in HG conditions (Supplementary Fig. 1B). NGF treatment at 100 ng/ml showed more prominent effects on cell differentiation than treatment at 10 ng/ml, as confirmed by MAP2 expression analysis (Supplementary Fig. 1C) and measurement of neurite outgrowth (Supplementary Fig. 1D and E). It should be noted that the neurite outgrowth in LG condition was significantly increased compared to that in HG condition (Supplementary Fig. 1E). Thus, to elucidate the interaction between glucose deprivation and NGF, both 10 ng/ml NGF and 100 ng/ml NGF were used in the following experiments. To examine the effects of glucose deprivation and NGF on SIRT1 expression, PC12 cells were cultured under the condition of either
LG-DMEM or HG-DMEM for 24–72 h. Expression levels of each protein were subsequently evaluated by western blotting analysis using specific antibodies. When cells were maintained in LGDMEM with 10 ng/ml NGF for 72 h, SIRT1 levels were significantly increased compared to when cells were maintained in HG-DMEM with 10 ng/ml NGF (Fig. 1A and B). The effect of NGF on this change was observed only in glucose-deprived conditions; 100 ng/ml NGF treatment significantly increased SIRT1 levels (Fig. 1A and B). On the other hand, 24–48 h of maintenance with LG-DMEM had no significant effect on SIRT1 levels either with or without high concentrations of NGF (data not shown). Real time PCR analysis showed that these changes in SIRT1 protein levels were correlated with SIRT1 gene expression (Fig. 1C). Furthermore, we analyzed whether intracellular localization of SIRT1 was affected in cells exposed to LG-DMEM or HG-DMEM for 72 h. In HG-DMEM conditions, the predominant SIRT1 localization was observed in cytosolic fractions, whereas in LG-DMEM conditions, most of the SIRT1 signal was observed in the nucleus (Fig. 1D and E). In addition, the nuclear localization of SIRT1 was significantly reduced by increasing NGF concentration from 10 ng/ml to 100 ng/ml (Fig. 1E). These experiments suggested that PC12 cells possessed glucose responsiveness, and glucose availability influenced both the level and localization of SIRT1. As we previously reported, regulation of FoxO3a and SIRT1 levels in C2C12 myotubes was similarly controlled by changes in
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Fig. 2. Effects of glucose and NGF on FoxO3a expression and localization in PC12 cells. (A–E) PC12 cells were differentiated under either LG or HG conditions in the presence of indicated amounts of NGF for 72 h. (A) Cell lysates were prepared as described in Section 2, and the same amounts of protein samples were subjected to western blotting using anti-FoxO3a antibody. (B) Densitometric analysis of (A) (*p < 0.05, n = 3, one-way ANOVA). (C) Total RNA were prepared and subjected to real time PCR analysis for evaluating FoxO3a gene expression (*p < 0.05, n = 3). (D) Differentiated PC12 cells under either LG or HG conditions were fixed and immunostained using anti-FoxO3a antibody (panel c, d, i, j). Hoechst 33342 staining was performed at the same time to confirm the position of the nucleus (panel a, b, g, h). Merged images were shown in panel e, f, k, l. All experiments were performed at least thrice, and similar results were obtained. (E) Signal intensity of nucleic FoxO3a and cytosolic FoxO3a in each panel were measured, and nuclear/cytoplasmic ratios were shown (**p < 0.01, n = 30–35, unpaired t-test).
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100 ng/ml NGF reversed this effect, suggesting that NGF prevents LG-induced FoxO3a accumulation in the nucleus (Fig. 2D and E). Finally, we examined the role of glucose deprivation-dependent SIRT1 induction in PC12 cells. Potent SIRT1 inhibitors, sirtinol or Ex527, were administered to PC12 cells cultured in either LG or HG conditions in the presence of 10 ng/ml NGF. Cell death was then evaluated by LDH assay. We observed that sirtinol or Ex527 had no apparent effect on cell death in HG conditions, but it significantly enhanced cell death in LG conditions (Fig. 3A and B). Cleaved caspase-3, which is often used as an index of apoptosis, was induced by glucose deprivation (Fig. 3C). This glucose deprivationinduced apoptosis, as assessed by measuring cleaved caspase-3 levels, was further potentiated by sirtinol treatment (Fig. 3C). These results suggested that even though glucose deprivation enhanced cell death, this condition also induced SIRT1 expression to protect cells from death (Fig. 3D).
4. Discussion
Fig. 3. Effects of SIRT1 inhibitor on PC12 cell death. (A–C) PC12 cells were differentiated under either LG conditions or HG conditions in the presence of 10 ng/ml NGF for 72 h. Indicated amounts of sirtinol (A and C) or Ex527 (B) were added to cell at 48 h. (A and B) Total cell death was measured by LDH assay (*p < 0.05, n = 3, oneway ANOVA). (C) The cell lysates were prepared, and apoptosis of PC12 cells was evaluated by measuring cleaved caspase-3 levels (*p < 0.05, n = 4, one-way ANOVA). (D) Schematic depiction of the present study. Reduced environmental glucose levels had biphasic effects: induction of cell death that was perhaps independent of FoxO3 regulation and reduction of cell death via SIRT1 induction.
glucose availability [10]. Therefore, we examined whether FOXO level in PC12 cells were affected by extracellular glucose deprivation. Similar to the SIRT1 results, FoxO3a levels were significantly increased in HG-DMEM with 10 ng/ml (Fig. 2A and B), which was accompanied by increased gene expression, as assessed by real time PCR analysis (Fig. 2C). Remarkably, the effects of NGF were unlike those for SIRT1, the addition of 100 ng/ml NGF in LG-DMEM significantly decreased FoxO3a level compared to the addition of 10 ng/ml NGF. (Fig. 2A and B). However, gene expression of FoxO3a was not influenced by NGF treatment (Fig. 2A and B). These results clearly indicated that increasing both glucose availability and NGF decreased FoxO3a expression, but the underlying mechanisms were completely different. Immunofluorescence studies revealed that glucose deprivation changed FoxO3a localization from the cytosol to the nucleus (Fig. 2D and E). Treatment with
Reduced glucose levels surrounding neurons can change the direction of their cellular fate towards death. This is an especially critical issue during ischemia. Our present findings strongly suggest that two glucose-sensitive proteins, SIRT1 and FoxO3a, are affected by reduced environmental glucose levels, which enhance their activities. This occurs even though the two proteins have opposite biological effects; SIRT1 promotes neuron survival [13,14], whereas FoxO3a promotes neuron apoptosis [15,16]. Intriguingly, NGF did not affect SIRT1 protein levels, but decreased FoxO3a protein levels, even though the impact of FoxO3a repression on cell death was minimal in PC12 cells. Overall, our results provide new insights in that glucose deprivation not only promotes cell death, but also exerts neuroprotective mechanisms by inducing SIRT1. Glucose deprivation is one of the major consequences of ischemia that subsequently induces neuronal cell death. The oxygen-glucose deprivation (OGD) model is widely used to investigate ischemia pathogenesis [20]. Certainly, the OGD model has an advantage for modeling ischemia, but distinguishing oxygen and glucose deprivation is necessary for understanding the detailed mechanisms of pathogenesis for these diseases. Besides, several reports have suggested abnormalities in brain glucose utilization in Alzheimer’s disease and amyotrophic lateral sclerosis [21–23]. Thus, our glucose deprivation model provides an important viewpoint on the pathogenesis of neuronal disorders. Liu Y et al. showed that glucose deprivation induced mitochondrial dysfunction and accumulation of reactive oxygen species (ROS), which promoted both necrosis and apoptosis in PC12 cells [24]. In this study, we confirmed that 48 h incubation of PC12 cells with LG-DMEM diminished glucose concentration in the medium to approximately zero, and initiated cell death, despite the presence of NGF (Supplementary Fig. 1A). Thus, glucose deprivation apparently increased PC12 cell death. Recent findings regarding SIRT1 have revealed that SIRT1 protein levels vary dramatically with nutrient availability in various tissues and cell lines [10,25,26]. On the other hand, whether this change is mediated by transcriptional control is still controversial [27,28]. In the present study, we observed SIRT1 gene expressional changes in response to glucose deprivation, which indicated changes that in glucose availability indeed regulates transcriptional properties for SIRT1 gene expression, at least in PC12 cells (Fig. 1C). In addition, we found that NGF treatment induced SIRT1 gene and protein expression, only in the absence of glucose (Fig. 1A and B). This finding is consistent with our previous observations that demonstrated interactions between glucose deprivation and insulin in C2C12 myocytes [10]. Overall, these interactions between extracellular glucose levels and growth factors
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affecting SIRT1 appear to be conserved among different types of cells. Interestingly, Sundaresan et al. [29] reported that SIRT1 deacetylates and activates Akt and PDK1, both of which are important molecules in growth factor signaling. This suggested that glucose deprivation- and growth factor-dependent SIRT1 induction involves a positive feedback mechanism to enhance growth factor signaling. Glucose deprivation also changed the intracellular localization of SIRT1—from cytosol to nucleus (Fig. 1D and E). The impact of this change in SIRT1 localization in PC12 cells is currently unknown; however, Jin et al. proposed that cytoplasm-localized SIRT1 enhances apoptosis [30], thus, nuclear-localized SIRT1 may function to prevent apoptosis in PC12 cells. It is well documented that induction of FoxO3a leads to ROS overproduction and stimulates apoptosis [31,32]. Moreover, it has also been established nucleic FoxO3a accumulation is directly involved in apoptosis [33,34]. However, the impact of reduction and locational changes of FoxO3a on cell viability appeared to be minimal, at least in PC12 cells because the cell death ratio was not significantly different between 10 ng/ml and 100 ng/ml NGF treatments. The biological roles of FoxO3a expression changes in PC12 cells are now under investigation. We found that inhibition of SIRT1 in the LG condition significantly enhanced cell death in PC12 cells. This result might not be surprising considering that resveratrol can inhibit betaamyloid–induced cell apoptosis through SIRT1 upregulation in PC12 cells [35]. However, our present results suggested that reduced environmental glucose levels had biphasic effects. Induction of cell death that was perhaps independent of FoxO3a regulation and the reduction of cell death via SIRT1 induction and nuclear translocation (Fig. 3D). The disturbance of this balance may directly determine neuronal cell fates. Acknowledgements We are deeply grateful to Dr. Shin-Ichiro Takahashi for PC12 cells. We also appreciate Dr. Masugi Nishihara for many constructive comments. This work was supported by Grants-in-Aid for Scientific Research (S) 23228004 and (C) 24580147 from the Japan Society for the Promotion of Science. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.neulet.2013.10.050. References [1] J. Rine, I. Herskowitz, Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae, Genetics 116 (1987) 9–22. [2] H.A. Tissenbaum, L. Guarente, Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans, Nature 410 (2001) 227–230. [3] L. Bordone, D. Cohen, A. Robinson, M.C. Motta, E. van Veen, A. Czopik, A.D. Steele, H. Crowe, S. Marmor, J. Luo, W. Gu, L. Guarente, SIRT1 transgenic mice show phenotypes resembling calorie restriction, Aging Cell 6 (2007) 759–767. [4] T. Finkel, C.X. Deng, R. Mostoslavsky, Recent progress in the biology and physiology of sirtuins, Nature 460 (2009) 587–591. [5] D. Herranz, M. Serrano, SIRT1: recent lessons from mouse models, Nat. Rev. Cancer 10 (2010) 819–823. [6] M.C. Haigis, D.A. Sinclair, Mammalian sirtuins: biological insights and disease relevance, Ann. Rev. Pathol. 5 (2010) 253–295. [7] M. Jiang, J. Wang, J. Fu, L. Du, H. Jeong, T. West, L. Xiang, Q. Peng, Z. Hou, H. Cai, T. Seredenina, N. Arbez, S. Zhu, K. Sommers, J. Qian, J. Zhang, S. Mori, X.W. Yang, K.L. Tamashiro, S. Aja, T.H. Moran, R. Luthi-Carter, B. Martin, S. Maudsley, M.P. Mattson, R.H. Cichewicz, C.A. Ross, D.M. Holtzman, D. Krainc, W. Duan, Neuroprotective role of Sirt1 in mammalian models of Huntington’s disease through activation of multiple Sirt1 targets, Nat. Med. 18 (2011) 153–158.
153
[8] M.E. Giannakou, L. Partridge, The interaction between FOXO and SIRT1: tipping the balance towards survival, Trends Cell Biol. 14 (2004) 408–412. [9] B.M. Burgering, G.J. Kops, Cell cycle and death control: long live Forkheads, Trends Biochem. Sci. 27 (2002) 352–360. [10] T. Nedachi, A. Kadotani, M. Ariga, H. Katagiri, M. Kanzaki, Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes, Am. J. Physiol. Endocrinol. Metab. 294 (2008) E668–E678. [11] A. Schurr, Energy metabolism, stress hormones and neural recovery from cerebral ischemia/hypoxia, Neurochem. Int. 41 (2002) 1–8. [12] B.C. White, J.M. Sullivan, D.J. DeGracia, B.J. O’Neil, R.W. Neumar, L.I. Grossman, J.A. Rafols, G.S. Krause, Brain ischemia and reperfusion: molecular mechanisms of neuronal injury, J. Neurol. Sci. 179 (2000) 1–33. [13] T. Araki, Y. Sasaki, J. Milbrandt, Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration, Science 305 (2004) 1010–1013. [14] B.L. Tang, SIRT1, neuronal cell survival and the insulin/IGF-1 aging paradox, Neurobiol. Aging 27 (2006) 501–505. [15] J. Gilley, P.J. Coffer, J. Ham, FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons, J. Cell Biol. 162 (2003) 613–622. [16] C. Barthélémy, C.E. Henderson, B. Pettmann, Foxo3a induces motoneuron death through the Fas pathway in cooperation with JNK, BMC Neurosci. 5 (2004) 48. [17] P.W. Gunning, G.E. Landreth, P. Layer, M. Ignatius, E.M. Shooter, Nerve growth factor-induced differentiation of PC12 cells: evaluation of changes in RNA and DNA metabolism, J. Neurosci. 1 (1981) 368–379. [18] R. Tabakman, P. Lazarovici, R. Kohen, Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia, J. Neurosci. Res. 68 (2002) 463–469. [19] G. Li, R. Ma, C. Huang, Q. Tang, Q. Fu, H. Liu, B. Hu, J. Xiang, Protective effect of erythropoietin on beta-amyloid-induced PC12 cell death through antioxidant mechanisms, Neurosci. Lett. 442 (2008) 143–147. [20] R. Tabakman, H. Jiang, I. Shahar, H. Arien-Zakay, R.A. Levine, P. Lazarovici, Neuroprotection by NGF in the PC12 in vitro OGD model: involvement of mitogen-activated protein kinases and gene expression, Ann. N.Y. Acad. Sci. 1053 (2005) 84–96. [21] S. Hoyer, Brain oxidative energy and related metabolism, neuronal stress, and Alzheimer’s disease: a speculative synthesis, J. Geriatr. Psychiatry Neurol. 6 (1993) 3–13. [22] R.J. Mark, Z. Pang, J.W. Geddes, K. Uchida, M.P. Mattson, Amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation, J. Neurosci. 17 (1997) 1046–1054. [23] M.C. Dalakas, J. Hatazawa, R.A. Brooks, G. Di Chiro, Lowered cerebral glucose utilization in amyotrophic lateral sclerosis, Ann. Neurol. 22 (1987) 580–586. [24] Y. Liu, X.D. Song, W. Liu, T.Y. Zhang, J. Zuo, Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line, J. Cell Mol. Med. 7 (2003) 49–53. [25] H.Y. Cohen, C. Miller, K.J. Bitterman, N.R. Wall, B. Hekking, B. Kessler, K.T. Howitz, M. Gorospe, R. de Cabo, D.A. Sinclair, Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase, Science 305 (2004) 390–392. [26] S. Kume, T. Uzu, K. Horiike, M. Chin-Kanasaki, K. Isshiki, S. Araki, T. Sugimoto, M. Haneda, A. Kashiwagi, D. Koya, Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney, J. Clin. Invest. 120 (2010) 1043–1055. [27] S. Nemoto, M.M. Fergusson, T. Finkel, Nutrient availability regulates SIRT1 through a forkhead-dependent pathway, Science 306 (2004) 2105–2108. [28] Y. Kanfi, V. Peshti, Y.M. Gozlan, M. Rathaus, R. Gil, H.Y. Cohen, Regulation of SIRT1 protein levels by nutrient availability, FEBS Lett. 582 (2008) 2417–2423. [29] N.R. Sundaresan, V.B. Pillai, D. Wolfgeher, S. Samant, P. Vasudevan, V. Parekh, H. Raghuraman, J.M. Cunningham, M. Gupta, M.P. Gupta, The deacetylase SIRT1 promotes membrane localization and activation of Akt and PDK1 during tumorigenesis and cardiac hypertrophy, Sci. Signal. 4 (2011) ra46. [30] Q. Jin, T. Yan, X. Ge, C. Sun, X. Shi, Q. Zhai, Cytoplasm-localized SIRT1 enhances apoptosis, J. Cell Physiol. 213 (2007) 88–97. [31] P. Obexer, K. Geiger, P.F. Ambros, B. Meister, M.J. Ausserlechner, FKHRL1mediated expression of Noxa and Bim induces apoptosis via the mitochondria in neuroblastoma cells, Cell Death Differ. 14 (2007) 534–547. [32] P. Obexer, J. Hagenbuchner, T. Unterkircher, N. Sachsenmaier, C. Seifarth, G. Böck, V. Porto, K. Geiger, M. Ausserlechner, Repression of BIRC5/survivin by FOXO3/FKHRL1 sensitizes human neuroblastoma cells to DNA damage-induced apoptosis, Mol. Biol. Cell 20 (2009) 2041–2048. [33] W.H. Zheng, S. Kar, R. Quirion, Insulin-like growth factor-1-induced phosphorylation of the forkhead family transcription factor FKHRL1 is mediated by Akt kinase in PC12 cells, J. Biol. Chem. 275 (2000) 39152–39158. [34] J. Chintapalli, S. Yang, D. Opawumi, S.R. Goyal, N. Shamsuddin, A. Malhotra, K. Reiss, L.G. Meggs, Inhibition of wild-type p66ShcA in mesangial cells prevents glycooxidant-dependent FOXO3a regulation and promotes the survival phenotype, Am. J. Physiol. Renal Physiol. 292 (2007) F523–F530. [35] X. Feng, N. Liang, D. Zhu, Q. Gao, L. Peng, H. Dong, Q. Yue, H. Liu, L. Bao, J. Zhang, J. Hao, Y. Gao, X. Yu, J. Sun, Resveratrol inhibits -amyloid-induced neuronal apoptosis through regulation of SIRT1-ROCK1 signaling pathway, PLoS One 8 (2013) e59888.
