epartment of Life Sciences, Graduate School of Life Sciences, Toyo University, Japan
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.
r t i c l e i n f o
rticle history:eceived 23 August 2013eceived in revised form 9 October 2013ccepted 20 October 2013
eywords:lucoseIRT1erve growth factor
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 sirtuin1 (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 proteinis not fully understood. In the present study, we examined whether extracellular glucose concentrationaffects the expression and localization of SIRT1 in PC12 cells. Further, we examined levels of forkhead boxO3a (FoxO3a), which is also controlled by changes in extracellular glucose concentration. We observed thetotal expression levels of SIRT1 and FoxO3a in PC12 cells were reduced when glucose availability increased
oxO3aC12
via gene expressional control, at least in part. Nuclear localization of SIRT1 and FoxO3a was increased byglucose deprivation. Even though the changes in extracellular glucose concentration regulated SIRT1 andFoxO3a in a similar direction, the effects of nerve growth factor on these two proteins were completelydifferent. 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 rolein protecting PC12 cells.
. Introduction
Recent evidence indicates that the glucose concentration sur-ounding cells is crucial for maintaining proper cellular functions.oth excess and deprivation of glucose can be detrimental to cells.herefore, an optimum glucose concentration is necessary to main-ain normal cellular functions. However, how cells monitor “an
ptimum amount of glucose” remains unclear.
Several key intracellular proteins that respond to glucosevailability were recently identified. One such protein, the
nicotinamide adenine dinucleotide (NAD)-dependent histonedeacetylase, silencing information regulator 2 (Sir2), was origi-nally identified in yeast and Caenorhabditis elegans [1,2]. Calorierestriction (or glucose deprivation) promotes Sir2 induction, whichresulted in an increased life span for that species. The mam-malian homologue of Sir2, sirtuins (SIRT), also responds to calorierestriction, although its impact on longevity is controversial [3–5].Moreover, recent studies have clearly demonstrated that the sub-strates of Sir2 or SIRT include histone, as well as other intracellularproteins such as, p53, PGC1a, Hif-1a, Hif-2a, HSF1, and FOXO1-4[6,7]. More importantly, the Sir2-dependent life span extensionobserved in C. elegans was dependent on Daf-16, a member offorkhead transcription factor O (FoxO) family [8]. Other impor-tant aspects of the FoxO family includes negative regulation bygrowth factor signaling, especially the phosphatidylinositol 3-
kinase (PI3K)-PKB/Akt cascade [9]. Akt phosphorylation of the FoxOfamily is considered the most predominant phenomenon in FoxOinactivation, followed by a change its localization from the nucleusto cytoplasm [9]. Overall, calorie restriction (or glucose deprivation)
ppears to cooperate with growth factor signaling, and therebyontrols SIRT1 and FoxO functions. In fact, we previously demon-trated that glucose availability defines the amounts of sirtuin 1SIRT1) and FoxO3a in C2C12 skeletal muscle cells [10]. Both theseroteins were induced in response to glucose deprivation via genexpressional changes. Furthermore, this mechanism appeared tonteract with the insulin-signaling cascade [10]. This type of glu-ose deprivation was observed in the central nervous system (CNS).or instance, blood supply restriction in brain ischemia resultedn hypoxia and glucose deprivation. The prognosis of ischemiaepends, in part, on the duration that the cells are exposed to oxy-en and glucose-free conditions and the presence of growth factorshat protect cells from death [11,12]. Whether SIRT1 and FOXO areegulated during this process in neuronal cells, in the same way as2C12 cells, remains elusive. This point of view could be importantince it has been reported SIRT1 promotes neuron survival [13,14],hereas FoxO3a promotes neuron apoptosis [15,16].
The rat pheochromocytoma (PC12) cells can be differentiatednto neurons with nerve growth factors [17], and are often usedor studying neuroprotection [18,19]. Therefore, in the presenttudy, we used PC12 cells to examine glucose-dependent regula-ion of SIRT1 and FOXO. Moreover, we examined whether the nerverowth factor (NGF) affects these regulating activities.
. Materials and methods
.1. Materials
The western blot detection kit (ECL plus or ECL primeetection reagents) was purchased from GE Healthcare Inc.Rockford, IL, USA). Dulbecco’s Modified Eagle Medium (DMEM),enicillin/streptomycin, and trypsin-EDTA were purchased fromakaraitesque (Kyoto, Japan). Cell culture equipment was obtained
rom BD Biosciences (San Jose, CA, USA). Calf Serum (CS) and Fetalovine Serum (FBS) were obtained from BioWest (Nuaille, France).
mmobilon-P was obtained from Millipore Corp. (Bedford, MA,SA). Unless otherwise noted, all chemicals were of the purestrade available from Nakaraitesque, Sigma Chemicals (St. Louis,O, USA) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
.2. Cell culture
PC12 cells were obtained through a generous gift from Dr. Shin-chiro Takahashi (The University of Tokyo, Tokyo, Japan). The cells
ere maintained in DMEM supplemented with 10% FBS, 30 �g/mlenicillin, and 100 �g/ml streptomycin (growth medium) at 37 ◦Cnder a 5% CO2 atmosphere. For biochemical studies, cells wererown on 6-well plates (Orange Scientific, Braine-l’Alleud, Belgium)t a density of 5 × 104 cells/well in 3 ml of growth medium, or on6-well plates (Orange Scientific) at a density of 5 × 103 cells/well
n 0.2 ml of growth medium. Three days after plating, cells typi-ally reached 50–70% confluence (Day 0). Differentiation was thennduced by switching the growth medium to DMEM containingither 5 mM glucose (LG-DMEM) or 22.5 mM glucose (HG-DMEM)upplemented with 10–100 ng/ml NGF, 30 �g/ml penicillin, and00 �g/ml streptomycin.
.3. Western blotting
The expression and phosphorylation of each protein were ana-yzed by western blot analysis using previously described methods10]. Detection of each protein was achieved with 1 h incuba-
ion with a 1:1000 dilution of primary antibody (anti-SIRT1,nti-FoxO3a, anti-cleaved caspase-3; Cell Signaling Technology,anvers, MA, USA). Specific total proteins were visualized after sub-
equent incubation with a 1:5000 dilution of anti-mouse or rabbit
ters 557 (2013) 148– 153 149
IgG conjugated to horseradish peroxidase and an ECL plus detec-tion procedure (GE Healthcare Inc.). Protein concentrations weredetermined using a bicinchoninic acid assay (BCA, Pierce Biotech.Inc.).
2.4. Immunofluorescence analysis
PC12 cells were cultured in the growth medium (DMEM/F-12supplemented with 10% FBS) for 3 days. The medium was thenswitched to experimental medium, and the cells were continuouslycultured for 72 h. The cells were then fixed with 4% PFA in PBS for15 min at room temperature (RT). Cells were washed twice withPBS (−) and incubated with 5% normal CS and 0.1% Triton X-100in PBS (blocking buffer) for 30 min, followed by incubation withprimary antibodies (anti-SIRT1; 1:200; Novocastra, Newcastle, UK;anti-FoxO3 antibody; 1:250; Cell Signaling Technology) for 2 h atRT. After washing the cells thrice with PBS, Alexa Fluor-conjugatedsecondary antibodies (Alexa Fluor 488 or 594; 1:500 dilution; Invi-trogen Corp.) and 5 �g/ml Hoechst 33258 in blocking buffer wereadded, and the cells were incubated for 1 h at RT. Again, cellswere washed thrice with PBS and then observed under LSM fivePascal/Axiovert 200 confocal microscopes (Carl Zeiss, Oberkochen,Germany). The images were analyzed by using fluorescence areaintensity measurements in the nucleus and in cytoplasm for SIRT1or FoxO3a. The average of nuclear/cytoplasmic ratios +/− SEM for10–35 cells was shown in the graph.
2.5. Glucose measurement
Glucose concentration in the cultured media were measuredusing a determiner GLE kit (Kyowa Medex, Tokyo, Japan).
2.6. Real time PCR
Fluorescence real time PCR analysis was performed usingStepOne instrument (Life Technologies Corporation, Grand Island,NY, USA) and SYBR Green detection kit according to the manu-facture’s procedure (Life Technologies or KAPA Biosystems Inc.,Woburn, MA, USA). PCR primers for measuring each gene includedthe 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 GGCCTC ATC TCA A-3′ and 5′-AGA TGG CGT GGG AGT CAC AA-3′; andGAPDH, 5′-GGC ACA GTC AAG GCT GAG AAT G-3′ and 5′-ATG GTGGTG AAG ACG CCA GTA-3′.
2.6.1. Measurement of cell deathPC12 cells were seeded on 96-well plates and differentiated as
described previously. The percentage of cell death was evaluatedusing the lactose dehydrogenase (LDH) plus kit (Roche DiagnosticsK.K., Basel, Switzerland) according to the manufacturer’s protocol.
2.7. Statistical analysis
Comparison among treatment groups was tested using one-wayANOVA and a post hoc Tukey test or Student’s t-test. Differences inwhich p < 0.05 were considered statistically significant.
3. Results
We previously reported the extracellular glucose levels defineSIRT1 and FoxO3a levels in C2C12 skeletal muscle cells [10]. There-fore, we initially examined whether glucose deprivation affects
SIRT1 and FoxO3 expression levels in PC12 cells. Before execut-ing the entire experiment, we evaluated glucose consumption. Inour experimental condition (see Section 2), cells were either grownin LG-DMEM or HG-DMEM; glucose concentration in these media
150 K. Fujino et al. / Neuroscience Letters 557 (2013) 148– 153
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 conditionsin 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 usinganti-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 forevaluating 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 immunostainedu the sas r resup –12,
w2ctaeN(Nsdt1ccsm((otiN
e
sing anti-SIRT1 antibody (panel c, d, i, j). Hoechst 33342 staining was performed athown in panel e, f, k, l. All experiments were performed at least thrice, and similaanel were measured, and nuclear/cytoplasmic ratios were shown (**p < 0.01, n = 10
as gradually decreased at an approximate rate of 0.4 g l−1 and4 h−1, which indicated LG-DMEM was completely deprived of glu-ose after 72 h of culture (Supplementary Fig. 1A). For elucidatinghe interaction between glucose deprivation and NGF, we initiallyttempted to compare between 0 ng/ml and 100 ng/ml NGF; how-ver, it was difficult to maintain cells for 24 h without serum orGF (data not shown). Thus, we decided to use 10 ng/ml NGF
presumably less effective concentration compared to 100 ng/mlGF) instead of 0 ng/ml NGF. To confirm if 10 ng/ml NGF was
uitable for following experiments, we evaluated the effects ofifferent concentration of NGF on cell viability and differentia-ion. PC12 cell death was indeed attenuated in the presence of0 ng/ml of NGF, as confirmed by the LDH assay, although theell death was significantly higher in LG conditions than in HGonditions (Supplementary Fig. 1B). NGF treatment at 100 ng/mlhowed more prominent effects on cell differentiation than treat-ent at 10 ng/ml, as confirmed by MAP2 expression analysis
Supplementary Fig. 1C) and measurement of neurite outgrowthSupplementary Fig. 1D and E). It should be noted that the neuriteutgrowth in LG condition was significantly increased compared tohat in HG condition (Supplementary Fig. 1E). Thus, to elucidate the
nteraction between glucose deprivation and NGF, both 10 ng/mlGF and 100 ng/ml NGF were used in the following experiments.
To examine the effects of glucose deprivation and NGF on SIRT1xpression, PC12 cells were cultured under the condition of either
me time to confirm the position of the nucleus (panel a, b, g, h). Merged images arelts were obtained. (E) Signal intensity of nucleic SIRT1 and cytosolic SIRT1 in eachunpaired t-test).
LG-DMEM or HG-DMEM for 24–72 h. Expression levels of eachprotein were subsequently evaluated by western blotting analysisusing specific antibodies. When cells were maintained in LG-DMEM with 10 ng/ml NGF for 72 h, SIRT1 levels were significantlyincreased compared to when cells were maintained in HG-DMEMwith 10 ng/ml NGF (Fig. 1A and B). The effect of NGF on this changewas observed only in glucose-deprived conditions; 100 ng/ml NGFtreatment significantly increased SIRT1 levels (Fig. 1A and B). Onthe other hand, 24–48 h of maintenance with LG-DMEM had nosignificant effect on SIRT1 levels either with or without high con-centrations of NGF (data not shown). Real time PCR analysis showedthat these changes in SIRT1 protein levels were correlated withSIRT1 gene expression (Fig. 1C). Furthermore, we analyzed whetherintracellular localization of SIRT1 was affected in cells exposed toLG-DMEM or HG-DMEM for 72 h. In HG-DMEM conditions, the pre-dominant SIRT1 localization was observed in cytosolic fractions,whereas in LG-DMEM conditions, most of the SIRT1 signal wasobserved in the nucleus (Fig. 1D and E). In addition, the nuclearlocalization of SIRT1 was significantly reduced by increasing NGFconcentration from 10 ng/ml to 100 ng/ml (Fig. 1E). These experi-ments suggested that PC12 cells possessed glucose responsiveness,
and glucose availability influenced both the level and localizationof SIRT1.
As we previously reported, regulation of FoxO3a and SIRT1 lev-els in C2C12 myotubes was similarly controlled by changes in
K. Fujino et al. / Neuroscience Letters 557 (2013) 148– 153 151
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 ofindicated 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 usinganti-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 evaluatingFoxO3a 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 (panelc, 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. Allexperiments were performed at least thrice, and similar results were obtained. (E) Signal intensity of nucleic FoxO3a and cytosolic FoxO3a in each panel were measured, andnuclear/cytoplasmic ratios were shown (**p < 0.01, n = 30–35, unpaired t-test).
152 K. Fujino et al. / Neuroscience Let
Fig. 3. Effects of SIRT1 inhibitor on PC12 cell death. (A–C) PC12 cells were differ-entiated under either LG conditions or HG conditions in the presence of 10 ng/mlNGF for 72 h. Indicated amounts of sirtinol (A and C) or Ex527 (B) were added to cellat 48 h. (A and B) Total cell death was measured by LDH assay (*p < 0.05, n = 3, one-way ANOVA). (C) The cell lysates were prepared, and apoptosis of PC12 cells wasevaluated by measuring cleaved caspase-3 levels (*p < 0.05, n = 4, one-way ANOVA).(hr
gltiatusoFTamif
D) Schematic depiction of the present study. Reduced environmental glucose levelsad biphasic effects: induction of cell death that was perhaps independent of FoxO3egulation and reduction of cell death via SIRT1 induction.
lucose availability [10]. Therefore, we examined whether FOXOevel in PC12 cells were affected by extracellular glucose depriva-ion. Similar to the SIRT1 results, FoxO3a levels were significantlyncreased in HG-DMEM with 10 ng/ml (Fig. 2A and B), which wasccompanied by increased gene expression, as assessed by realime PCR analysis (Fig. 2C). Remarkably, the effects of NGF werenlike those for SIRT1, the addition of 100 ng/ml NGF in LG-DMEMignificantly decreased FoxO3a level compared to the additionf 10 ng/ml NGF. (Fig. 2A and B). However, gene expression ofoxO3a was not influenced by NGF treatment (Fig. 2A and B).hese results clearly indicated that increasing both glucose avail-
bility and NGF decreased FoxO3a expression, but the underlyingechanisms were completely different. Immunofluorescence stud-
es revealed that glucose deprivation changed FoxO3a localizationrom the cytosol to the nucleus (Fig. 2D and E). Treatment with
ters 557 (2013) 148– 153
100 ng/ml NGF reversed this effect, suggesting that NGF preventsLG-induced FoxO3a accumulation in the nucleus (Fig. 2D and E).
Finally, we examined the role of glucose deprivation-dependentSIRT1 induction in PC12 cells. Potent SIRT1 inhibitors, sirtinol orEx527, were administered to PC12 cells cultured in either LG orHG conditions in the presence of 10 ng/ml NGF. Cell death wasthen evaluated by LDH assay. We observed that sirtinol or Ex527had no apparent effect on cell death in HG conditions, but it sig-nificantly enhanced cell death in LG conditions (Fig. 3A and B).Cleaved caspase-3, which is often used as an index of apoptosis, wasinduced by glucose deprivation (Fig. 3C). This glucose deprivation-induced apoptosis, as assessed by measuring cleaved caspase-3levels, was further potentiated by sirtinol treatment (Fig. 3C). Theseresults suggested that even though glucose deprivation enhancedcell death, this condition also induced SIRT1 expression to protectcells from death (Fig. 3D).
4. Discussion
Reduced glucose levels surrounding neurons can change thedirection of their cellular fate towards death. This is an especiallycritical issue during ischemia. Our present findings strongly sug-gest that two glucose-sensitive proteins, SIRT1 and FoxO3a, areaffected by reduced environmental glucose levels, which enhancetheir activities. This occurs even though the two proteins haveopposite 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 pro-tein levels, even though the impact of FoxO3a repression on celldeath was minimal in PC12 cells. Overall, our results provide newinsights 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 ofischemia that subsequently induces neuronal cell death. Theoxygen-glucose deprivation (OGD) model is widely used to inves-tigate ischemia pathogenesis [20]. Certainly, the OGD model has anadvantage for modeling ischemia, but distinguishing oxygen andglucose deprivation is necessary for understanding the detailedmechanisms of pathogenesis for these diseases. Besides, severalreports have suggested abnormalities in brain glucose utilizationin Alzheimer’s disease and amyotrophic lateral sclerosis [21–23].Thus, our glucose deprivation model provides an important view-point on the pathogenesis of neuronal disorders. Liu Y et al. showedthat glucose deprivation induced mitochondrial dysfunction andaccumulation of reactive oxygen species (ROS), which promotedboth necrosis and apoptosis in PC12 cells [24]. In this study, weconfirmed that 48 h incubation of PC12 cells with LG-DMEM dimin-ished glucose concentration in the medium to approximately zero,and initiated cell death, despite the presence of NGF (Supplemen-tary Fig. 1A). Thus, glucose deprivation apparently increased PC12cell death.
Recent findings regarding SIRT1 have revealed that SIRT1 pro-tein levels vary dramatically with nutrient availability in varioustissues and cell lines [10,25,26]. On the other hand, whether thischange is mediated by transcriptional control is still controversial[27,28]. In the present study, we observed SIRT1 gene expressionalchanges in response to glucose deprivation, which indicatedchanges that in glucose availability indeed regulates transcrip-tional properties for SIRT1 gene expression, at least in PC12 cells(Fig. 1C). In addition, we found that NGF treatment induced SIRT1gene and protein expression, only in the absence of glucose (Fig. 1A
and B). This finding is consistent with our previous observationsthat demonstrated interactions between glucose deprivationand insulin in C2C12 myocytes [10]. Overall, these interac-tions between extracellular glucose levels and growth factors
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ffecting SIRT1 appear to be conserved among different typesf cells. Interestingly, Sundaresan et al. [29] reported that SIRT1eacetylates and activates Akt and PDK1, both of which are impor-ant molecules in growth factor signaling. This suggested thatlucose deprivation- and growth factor-dependent SIRT1 induc-ion involves a positive feedback mechanism to enhance growthactor signaling. Glucose deprivation also changed the intracellularocalization of SIRT1—from cytosol to nucleus (Fig. 1D and E). Thempact of this change in SIRT1 localization in PC12 cells is currentlynknown; however, Jin et al. proposed that cytoplasm-localizedIRT1 enhances apoptosis [30], thus, nuclear-localized SIRT1 mayunction to prevent apoptosis in PC12 cells.
It is well documented that induction of FoxO3a leads to ROSverproduction and stimulates apoptosis [31,32]. Moreover, it haslso been established nucleic FoxO3a accumulation is directlynvolved in apoptosis [33,34]. However, the impact of reductionnd locational changes of FoxO3a on cell viability appeared to beinimal, at least in PC12 cells because the cell death ratio was not
ignificantly different between 10 ng/ml and 100 ng/ml NGF treat-ents. The biological roles of FoxO3a expression changes in PC12
ells are now under investigation.We found that inhibition of SIRT1 in the LG condition sig-
ificantly enhanced cell death in PC12 cells. This result mightot be surprising considering that resveratrol can inhibit beta-myloid–induced cell apoptosis through SIRT1 upregulation inC12 cells [35]. However, our present results suggested thateduced environmental glucose levels had biphasic effects. Induc-ion of cell death that was perhaps independent of FoxO3aegulation and the reduction of cell death via SIRT1 induction anduclear translocation (Fig. 3D). The disturbance of this balance mayirectly determine neuronal cell fates.
cknowledgements
We are deeply grateful to Dr. Shin-Ichiro Takahashi for PC12ells. We also appreciate Dr. Masugi Nishihara for many construc-ive comments. This work was supported by Grants-in-Aid forcientific Research (S) 23228004 and (C) 24580147 from the Japanociety for the Promotion of Science.
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.neulet.2013.10.050.
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