Metformin induced protection against oxidative stress is associated with AKT/mTOR restoration in PC12 cells Behzad Khallaghi, Fatemeh Safarian, Sanaz Nasoohi, Abolhassan Ahmadiani, Leila Dargahi PII: DOI: Reference:
S0024-3205(16)30073-X doi: 10.1016/j.lfs.2016.02.024 LFS 14709
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
31 May 2015 26 January 2016 7 February 2016
Please cite this article as: Khallaghi Behzad, Safarian Fatemeh, Nasoohi Sanaz, Ahmadiani Abolhassan, Dargahi Leila, Metformin induced protection against oxidative stress is associated with AKT/mTOR restoration in PC12 cells, Life Sciences (2016), doi: 10.1016/j.lfs.2016.02.024
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ACCEPTED MANUSCRIPT Metformin induced protection against oxidative stress is associated with AKT/mTOR restoration in PC12 cells
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Behzad Khallaghi1, Fatemeh Safarian1, Sanaz Nasoohi2, Abolhassan Ahmadiani1, 3, Leila
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Dargahi4* 1
Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Department of Pharmacology and Toxicology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 3Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia; 4NeuroBiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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Behzad Khallaghi and Fatemeh Safarian, as co-first author, contributed equally to this work.
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*Corresponding author Leila Dargahi NeuroBiology Research Center, Shahid Beheshti University of Medical sciences, Tehran, Iran. Email Address: [email protected] P. O. BOX: 19615-1178 Telephone: +98-21-22429768-9 Fax: +98-21-22432047
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ACCEPTED MANUSCRIPT Abstract
Aims:
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Reactive oxygen species have been recognized to impair cell function through suppressing Akt
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the well-known pro-survival molecule. Pile of concrete evidence imply metformin as an Insuline sensitizer may enhance Akt/mTOR activity however the significance of Akt/mTOR recruitment
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has not yet been revealed in metformin induced neuroprotection against oxidative stress. Main methods:
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In the current study using H2O2 induced injury in PC12 cells; we first examined metformin impact on cell death by MTT assay and visual assessment. Metformin pretreated cells were then
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subjected to immunoblotting as well as real time PCR to find PI3K, Akt, mTOR and S6K concurrent transcriptional and post-transcriptional changes. The proportions of phosphorylated to non-phosphorylated constituents of PI3K/Akt/mTOR/S6K were determined to address their
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activation upon metformin treatment. Key findings:
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According to cells morphology and MTT data metformin led to significant protection against H2O2 induced injury in 0.1 and 0.5 mM concentrations. Metformin induced protection concurred
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with elevated PI3K/Akt/mTOR/S6K activity as well as enhanced GSH levels. These changes paralleled with a profound decline in the corresponding transcripts as determined by rea time
Significance:
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PCR.
Taken together our experimentation support the hypothesis that Akt/mTOR/S6K cascade may contribute to metformin alleviating effect. The present work while highlighting metformin antioxidant characteristics, concludes that Akt/mTOR signaling might be central to the drug’s alleviating effects. Keywords: Metformin; Oxidative stress; Akt; mTOR
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1. Introduction
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Metformin has been introduced as a neuroprotective agent by in vitro examinations [1] as
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well as in vivo experiments describing pro-survival and anti-inflammatory impacts of metformin in Huntington's disease and multiple sclerosis experimental models [2, 3]. The pro-survival
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effects of metformin has been ascribed to the decreased intracellular production of reactive oxygen species (ROS) [4] and to the improved balance between pro-oxidants and antioxidants
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[5].
Empirically metformin has been also shown to stimulate mammalian target of rapamycin
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(mTOR) especially in low doses [6] in which it exerts significant cytoprotection in non-cancerous cells [7, 8]. mTOR regulates a variety of cellular responses to extracellular signals particularly nutrients availability and stress [9]. This is predominantly mediated through its best-studied
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down-stream effectors S6K1 (p70 ribosomal protein S6 kinase 1) and 4EBP1 (eIF4E binding protein). The serine/threonine protein kinase Akt, a downstream effector of PI3K, is a critical
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mediator of mTOR activity [10]. The prominence of Akt/mTOR integrity have been documented by several empirical evidences to promote cell survival [11, 12] and proliferation which is mainly
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mediated through S6K activation. In line with this, there are pile of concrete evidence indicating that Akt/mTOR inhibitors like Rapamycine may be regarded as therapeutic agents to impede
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cancerous proliferation of different cell types [13, 14]. Notably, ROS such as superoxide (O2−) and hydrogen peroxide (H2O2) could also regulate tyrosine phosphorylation [15, 16] and may suppress mTOR activity through Akt inhibition [17]. Nevertheless, there are also recent evidences implying ROS-induced repression of mTOR may be downstream to tuberous sclerosis complex (TSC) signaling node on the peroxisome which functions as a cellular sensor for ROS to regulate mTOR and then autophagy [18]. Taken together, Akt/mTOR signaling seems to get remarkably affected by both oxidative stress and metformin. Therefore, theoretically it could be assumed that Akt/Mtor may mediate anti-oxidant effects of the drug. Accordingly, in the present work we aimed to determine the correlation between Akt/mTOR activity and metformin induced protection against oxidative stress. Since the H2O2 induced insult was associated with dampened Akt/mTOR, the main purpose was to find whether metformin protective doses enhance Akt/mTOR activity 3
ACCEPTED MANUSCRIPT simultaneously. To this end PC12 cells as commonly used neural like cells were exposed to H2O2 in IC50 concentrations and effect of metformin pretreatment was determined at transcriptional and post-translational levels of PI3K/Akt/mTOR/S6K signaling components. The
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concurrent changes in antioxidants’ status; catalase (CAT) and superoxide dismutase (SOD)
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activity and glutathione (GSH) levels, were also evaluated in stressed cells exposed to protective concentrations of metformin that has been identified as an anti-oxidant agent [19].
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2. Experimental Procedures 2.1. Cell culture and treatment
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PC12 cells (National cell bank, Pasteur institute of Iran) were maintained in high glucose (4500 mg/L) DMEM containing heat inactivated fetal bovine serum (FBS) (10% v/v), Penicillin
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(100 units/ml) and Streptomycin (100 µg/ml) (all from Invitrogen) at 37 °C in 95% air and 5% carbon dioxide. PC12 cells, at passage number 8-10, were seeded in 96-well plates (JET BioFil)
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at a density of 1×104 cells per well in serum free DMEM medium (Invitrogen) and were allowed to attach and expand for 24 h. H2O2 at the concentration of 150 µM was used to induce oxidative
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stress in PC12 cells. Pre-treatment with varying concentrations of freshly prepared Metformin (0.1, 0.5, 1, 3, 5, 7 mM) solution was performed for 60 min prior to stress induction by H2O2.
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Cells were then subjected to experimental analyses following H2O2 exposure for 24 h. A negative control cell group containing culture medium alone was also included as reference cells.
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2.3. Cell survival assay
Cell viability was grossly assessed by the stress related changes as determined in three separated fiedls in each photomicrographs. The mitochondrial integrity was also evaluated by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction to purple formazan [20]. Briefly, cells were incubated with MTT solution (final concentration 0.1-7 mg/mL) for 3 h at 37 °C. The medium was carefully removed by aspiration and formazan crystals were dissolved in 100 µl of dimethyl sulfoxide (DMSO). The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm and was reported as the percentage of cell viability compared to control.
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ACCEPTED MANUSCRIPT 2.4. Superoxide dismutase activity assay Superoxide dismutase (SOD) activity was analyzed by the methods of Kakkar et al [21]. Briefly the assay mixture containing appropriate amounts of nicotinamide adenine dinucleotide,
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phenazine methosulphate and nitroblue tetrazolium (750, 186 and 300 µM respectively) were
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subjected to react with SOD present in cellular lysate. Following adequate incubation in 30 οC (90 sec), the mixture was stirred after n-butanol addition. The measured absorbance at 560 nm
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was considered as the color intensity of amino blue tetrazolium formazan (ABTF), the chromogen in butanol. Samples which resulted in 50% inhibition of total nitroblue tetrazolium
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reduction to ABTF per each mg of protein, was considered to possess one unit of enzymatic activity.
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2.5. Catalase activity assay
Catalase (CAT) activity was determined by the method of Aebi [22]. Simply cell lysate was
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mixed with phosphate buffer (50mM, pH 7.0) and subjected to react with the freshly prepared H2O2 (30mM). The rate of decomposition of H2O2 measured by the mixture’s absorbance at
2.6. GSH level analysis
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240 nm was considered as the CAT activity.
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Total GSH levels were measured in whole cell lysates according to the rate of colorimetric change of 5,5′-dithiobis-2-nitrobenzoic acid (Ellman’s reagent) as determined by the
µmol/mg.
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spectrophotometric method and absorbance at 405 nm [23]. GSH concentration was expressed as
2.7. Immunoblot Analysis The cells were washed with PBS scraped in ice-cold RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% sodium deoxycholate, 1% NP-40, 0.5 mM PMSF), and incubated on ice for 30 minutes. The cellular debris was removed by centrifugation (8000g for 15 min) at 4 °C and the cell lysate was carefully transferred to the microcentrifuge tubes. Proteins in samples standardized by Bradford assay [24], were separated by SDS electrophoresis and transferred to PVDF membranes. To estimate the amount of target proteins, membranes were then incubated with specific primary antibodies against Akt1/2/3/3 (H-136) and p-Akt1/2/3 (Ser 473), PI3 Kinase Class III, phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199), mTOR, phospho5
ACCEPTED MANUSCRIPT mTOR (Ser248), p70S6 Kinase and phospho-p70 S6 Kinase (Thr389). Antibodies were obtained from Cell Signaling Technology and prepared in 1:1000 v/v dilutions except for Akt1 and p-Akt1 which were purchased from Santa Cruz and used in 1/600 v/v dilution. To visualize protein
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bands, the blots were incubated with horseradish peroxidase-linked secondary antibody (#7074)
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(1:10000 v/v; Cell Signaling Technology) which is directly detectable by chemiluminescence kit reagent (Amersham). The attained scans were then analyzed semi-quantitatively using Image J
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software in proportion to β-actin band intensity as internal control. 2.8. Quantitative Real-Time PCR
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Total RNA was extracted from cultured cells using NucleoSpin II kit (Macherey-Nagel GmbH & Co KG) and cDNA was synthesized using Revert Aid first strand cDNA synthesis kit
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(Thermo Fisher Scientific Inc.), all according to the manufacturers’ instructions. Quantitative RTPCR was performed on an ABI 7500 real time PCR system (PE Applied Biosystems, Foster City, CA, USA) using Maxima SYBR Green/ROX qPCR Master Mix according to the manufacturer’s
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recommendations with primer pairs listed in Table 1. β-actin was used as the reference housekeeping gene. The relative expression of mRNA transcripts, standard error means and p-
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values for differences in fold-expression were determined by the relative expression ratio method
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via the Relative Expression Software Tool-Multiple Condition Solver (REST-MCS) [25]. 2.9. Statistical Analysis
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All results represent the mean value from at least three separate experiments each with at least two replicates. All the data with the exception of those obtained at real-time PCR, were statistically analyzed using SPSS software. Normality of data was verified using the ShapiroWilk test. Analysis was performed with one-way ANOVA followed by Tukey post-hoc test and a P-value < 0.05 was considered as significant. Data are presented as mean ±SEM with the exception of relative fold changes in gene expression data which are presented as mean ±SD.
3. Results
3.1. Metformin does not exert dose-dependent protection against H2O2-induced injury in PC-12 cell
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ACCEPTED MANUSCRIPT According to the presumptive diverse roles of metformin as a pro-survival [1] or proapoptotic [26] agent and for the differences in the experimental setup (eg. different cell lines) a dose response assay was primarily performed to determine the protective metformin
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concentrations. Photomicrographs show that the presence of metformin together with H2O2
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attenuates morphological alterations caused by H2O2 as defined by the ratio of stressed round shaped cells (Fig. 1a,b). Nevertheless, such impact was not detectable in metformin
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concentrations higher than 0.5 mM. Similarly, metformin (at 5 discreet concentrations ranging from 0.1 to 5 mM) did not show consistent dose-dependent responses in MTT assay. That is
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while incubation of the PC12 cells with metformin at 1, 3, and 5 mM concentrations did not result in restoring cell viability, metformin exposure at 0.1 and 0.5 mM concentrations rendered
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PC12 cells nearly resistant to H2O2 (Fig. 1c). Given metformin alone (at 5 discrete concentrations ranging from 0.1 to 5 mM) did not change MTT assay results (data not shown), such alterations are not seemingly due to metformin impact on intact cells. H2O2 concentration
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used to cause oxidative stress (150 µM) was selected according to the previously reported H2O2
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LD50% of 150 µM in a 24 h period of exposure as identified by our previous data [27].
3.2. The H2O2 induced decline in CAT and GSH was reversed by metformin
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To evaluate cells’ antioxidant strength, metformin effects was assessed on SOD, CAT and GSH as major detoxifying agents which degrade superoxide and H2O2 [28, 29]. According to the
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obtained data the antioxidant activity of SOD and CAT and the content of GSH decreased by H2O2 (150 µM) treatment in PC12 cells with higher confidence interval for CAT. Metformin treatment was not associated with any change in H2O2 induced escalation in SOD activity. Instead, as obtained by CAT and GSH assessment, PC12 cells exhibited improved CAT and GSH while pretreated with metformin low concentrations. Noteworthy, the improving effect of metformin on CAT and GSH vanished in doses higher than 1 mM and 0.5 mM respectively (data not shown). This may be corroborated with our MTT data in which metformin could not preserve cells’ viability while applied in high concentrations. Thus in order to evaluate the molecular alterations concurrent with metformin alleviating effects we proceeded to next experiments with metformin at 0.1, 0.5 and 1mM dosages (Fig 2).
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ACCEPTED MANUSCRIPT 3.3. Protective effects of metformin concurred with enhanced PI3K/Akt/mTOR/S6K activity Impact of oxidative stress on PI3K/Akt/mTOR/S6K may be extensively context sensitive
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[17]. Several concrete evidences suggest ROS may suppress PI3K/S6K while others imply in
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pro-survival concentrations, ROS might activate PI3K/S6K ROS [30, 31]. According to our data, in our set of experiments H2O2 effectively inhibited PI3K/S6K activation as determined by the
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proportion of phosphorylated to un-phosphorylated PI3K/S6K constituents and the corresponding down streams mTOR and S6K. The relatively coordinated changes in PI3K, Akt, mTOR and
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S6K phosphorylation was suggestive of profound impression by H202. Notably, metformin augmented PI3K/S6K activity in the low dosages (0.1 and 0.5 mM) while in 1 mM it left the
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H2O2 induced PI3K/S6K repression almost entirely unaffected or even slightly intensified. As presented in figure 3, nearly all corresponding changes due to 1 mM metformin share the property of showing significant decline compared to control while it is not the case for the lower
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concentrations (0.1 and 0.5 mM). Taken together with the MTT data, this may imply the remarkable pro-survival effect of metformin was almost coincident with the restoring PI3K/S6K
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pathway.
proteins activation
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3.4. Changes in PI3K/Akt/mTOR/S6K mRNA transcripts inversely correlate with that of
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Quantitative real time PCR results of three independent experiments showed there is a significant shift towards enhanced transcription of PI3K/Akt/mTOR/S6K cascade proteins with increasing dosages of metformin (0.1 to 1 mM). As shown in figure 4, seemingly the upward change of transcripts in 1 mM concentration of metformin is the only case of over-expression that was strong enough to markedly surpass the corresponding one in control that is determined by 1 in y axis. The relevant refinements were also performed in the legend and also the pertinent result section.The maximal mRNA transcript was detected in 1 mM which surpassed that of control
cells. Interestingly as a common future, metformin in all included doses (0.1, 0.5 and 1 mM) seemingly reversed the proportion of Akt and mTOR transcripts to the up-stream effector PI3K. Nevertheless, here we could not suggest any correlation with protective effects of metformin since the mRNA levels did not consistently exceed that of control group in both protective concentrations of metformin (i.e.; 0.1 and 0.5 Mm) (Fig4).
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ACCEPTED MANUSCRIPT 4. Discussion
Metformin antioxidant properties have been shown to reserve endothelial function [32] and
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protect against cardiovascular [33] and central [34] complications in diabetic subjects, however;
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no evidence has been reported considering PI3K/S6K implication. Results of the present work demonstrate that PI3K/S6K activity is restored during metformin induced protection. In this
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connection, our data suggest metformin may enhance cell survival in the face of oxidative stress at least partly through strengthening antioxidant systems particularly GSH and CAT.
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Basically, mitochondrion is the primary target of metformin within the cell in which metformin transiently inhibits complex I in electron transport chain. In the view of diabetes
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therapy, this results in a drop in energy charge [35] and consequently in AMP/ATP proportion which in turn activates AMP-activated protein kinase (AMPK) which negatively regulates mTOR/ S6K activation [36, 37]. On the other side, the same mechanism may also account for the
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hampered ROS production in reverse-electron flux at respiratory-chain complex 1 by metformin [38]. Besides reducing ROS levels, metformin administration may also end with strengthened
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antioxidant systems [39, 40] which might contribute to the documented nephro-protective effects of metformin in diabetes mellitus [41].
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According to our findings, low dose metformin was efficient enough to retrieve CAT and GSH despite lack of SOD restoration. This is somehow verified by previous works describing
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metformin may enhance CAT activity and GSH levels [42, 43, 44], while there is not enough evidence supporting SOD augmentation by metformin [45]. Given that oxidative stress regulates the activity of the cell survival factor Akt [35] we sought to correlate Akt/mTOR cascade to H2O2 toxicity. Having in mind H2O2 impact on Akt activity may strictly depend on cell type as well as exposure conditions [17], our results described a marked decrease in phosphorylation of Akt/mTOR inactive constituents in H2O2 treated PC12 cells. This is in agreement with previous studies indicating H2O2 induces a drop in Akt kinase activity in PC12 cells [46] which may hypothetically take part in the induced injury [47, 48]. As an effector downstream to Insulin receptor, PI3K may get activated upon Insulin binding. Metformin is empirically considered as an Insulin sensitizer in spite that it is devoid of metabolites or conjugates that could activate Insulin receptors [46]. This is noteworthy that Insulin sensitizing property of metformin has been supported by in vitro [50] and in vivo [51] 9
ACCEPTED MANUSCRIPT evidences implying metformin enhances insulin function by improving Insulin receptors-linked tyrosine phosphorylation. In line with this, low dose metformin has been shown to affect neural injury induced by glucose-oxygen deprivation through modulating phosphorylation of β-subunit
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tyrosine in Insulin receptor [52].
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Our data also described a transient PI3K/S6K signaling activation in the low dose (0.1 or 0.5 mM) while it was coincident with significant protection. This primary PI3K/S6K augmentation
Akt/mTOR activation by low dose metformin [52].
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may be the result of neutralizing H2O2 effects [46] or more probably, the direct consequent of
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Unexpectedly, our data shows the slightly higher dose of metformin (1 mM) did not keep on reversing PI3K/S6K activity against H2O2 toxicity. Actually, in some cases PI3K/S6K
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suppression even enhanced in this concentration. Such intensified PI3K/S6K suppression might be ascribed to switching between downstream effectors. That is based on empirical evidences, metformin may reasonably suppress PI3K/S6K activity by AMPK stimulation following
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interfering with the mitochondrial electron transport as described before. This way, activated AMPK switches cells from an anabolic to a catabolic state, thus shutting down the ATP-
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consuming proliferative pathways like mTOR/S6K signaling [53]. The observed S6K suppression by metformin increasing doses may indicate that such interference with Akt/S6K
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signaling may commence even in very low concentrations of the drug. Noteworthy, AMPK also has been shown to exert pro-survival effects in various cell types
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[54, 55], but here we can’t add anything about AMPK involvement as we did not examine AMPK activity. However, this presumptive AMPK stimulation by metformin might explain the opposite changes observed in PI3K/S6K by metformin at relatively higher concentration of 1 Mm. Intriguingly, in contrary to post translational effects of metformin, our results on mRNA assay identified that the transcriptional levels of PI3K/S6K signaling molecules were enhanced by any enhancement in metformin exposure. There are concrete evidences implying downstream components like mTOR may negatively control the activity of Akt/mTOR signaling [56, 57]. In this view, the initially inversed PI3K/S6K transcription at metformin low concentrations may be accountable for the transient augmentation of Akt/mTOR signaling by the low concentration of metformin. Nonetheless the reverse correlation between transcriptional and translational levels of
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ACCEPTED MANUSCRIPT PI3K/S6K constituents might be suggestive of a negative feedback loop, we did not find any
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evidence implying relevant gene transcripts are regulated by activated Akt or PI3K.
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5. Conclusion
Collectively, the present work shows metformin induced protection in PC12 cells is associated with Akt/mTOR restoration however using kinase inhibitors would substantially
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contribute to verify Akt/mTOR implication. While the absence of pro-survival effect of metformin was coincident with dismissing GSH elevation, it needs to be unraveled whether these
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anti-oxidants properties is a direct consequent of PI3K/S6K activity. Lastly regarding probable differences between the relevant intracellular cascades, examining alternative pathways and in
neuroprotective effects of the drug.
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Acknowledgements
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particular considering virtual neural cultures, may shed may shed light on literally
This study was supported financially by Neuroscience Research Center, Shahid Beheshti
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University of Medical Sciences. The authors thank Dr. Khodagholi F for valuable guidance on
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Figure legends Fig. 1. Effect of metformin on H2O2 induced cell injury as determined by MTT assay and morphological alterations. A 24 h period of H2o2 (150 µM) exposure caused a significant decline in PC12 cells viability. The representative photomicrographs of PC12 cells (a) demonstrate the corresponding substantial decline in round forms (b) due to protection induced by metformin at 0.1 and 0.5 mM concentrations. In the same concentrations MTT assay data (b) agrees the antioxidant activity of metformin. Values are expressed as mean ± SEM from three independent experiments, each of which including four replicates. *p < 0.05, **p < 0.01, ***p < 0.001 versus control; #p
S0024-3205(16)30073-X doi: 10.1016/j.lfs.2016.02.024 LFS 14709
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Received date: Revised date: Accepted date:
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Please cite this article as: Khallaghi Behzad, Safarian Fatemeh, Nasoohi Sanaz, Ahmadiani Abolhassan, Dargahi Leila, Metformin induced protection against oxidative stress is associated with AKT/mTOR restoration in PC12 cells, Life Sciences (2016), doi: 10.1016/j.lfs.2016.02.024
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ACCEPTED MANUSCRIPT Metformin induced protection against oxidative stress is associated with AKT/mTOR restoration in PC12 cells
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Behzad Khallaghi1, Fatemeh Safarian1, Sanaz Nasoohi2, Abolhassan Ahmadiani1, 3, Leila
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Dargahi4* 1
Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Department of Pharmacology and Toxicology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 3Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia; 4NeuroBiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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Behzad Khallaghi and Fatemeh Safarian, as co-first author, contributed equally to this work.
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*Corresponding author Leila Dargahi NeuroBiology Research Center, Shahid Beheshti University of Medical sciences, Tehran, Iran. Email Address: [email protected] P. O. BOX: 19615-1178 Telephone: +98-21-22429768-9 Fax: +98-21-22432047
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ACCEPTED MANUSCRIPT Abstract
Aims:
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Reactive oxygen species have been recognized to impair cell function through suppressing Akt
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the well-known pro-survival molecule. Pile of concrete evidence imply metformin as an Insuline sensitizer may enhance Akt/mTOR activity however the significance of Akt/mTOR recruitment
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has not yet been revealed in metformin induced neuroprotection against oxidative stress. Main methods:
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In the current study using H2O2 induced injury in PC12 cells; we first examined metformin impact on cell death by MTT assay and visual assessment. Metformin pretreated cells were then
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subjected to immunoblotting as well as real time PCR to find PI3K, Akt, mTOR and S6K concurrent transcriptional and post-transcriptional changes. The proportions of phosphorylated to non-phosphorylated constituents of PI3K/Akt/mTOR/S6K were determined to address their
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activation upon metformin treatment. Key findings:
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According to cells morphology and MTT data metformin led to significant protection against H2O2 induced injury in 0.1 and 0.5 mM concentrations. Metformin induced protection concurred
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with elevated PI3K/Akt/mTOR/S6K activity as well as enhanced GSH levels. These changes paralleled with a profound decline in the corresponding transcripts as determined by rea time
Significance:
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PCR.
Taken together our experimentation support the hypothesis that Akt/mTOR/S6K cascade may contribute to metformin alleviating effect. The present work while highlighting metformin antioxidant characteristics, concludes that Akt/mTOR signaling might be central to the drug’s alleviating effects. Keywords: Metformin; Oxidative stress; Akt; mTOR
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1. Introduction
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Metformin has been introduced as a neuroprotective agent by in vitro examinations [1] as
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well as in vivo experiments describing pro-survival and anti-inflammatory impacts of metformin in Huntington's disease and multiple sclerosis experimental models [2, 3]. The pro-survival
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effects of metformin has been ascribed to the decreased intracellular production of reactive oxygen species (ROS) [4] and to the improved balance between pro-oxidants and antioxidants
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[5].
Empirically metformin has been also shown to stimulate mammalian target of rapamycin
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(mTOR) especially in low doses [6] in which it exerts significant cytoprotection in non-cancerous cells [7, 8]. mTOR regulates a variety of cellular responses to extracellular signals particularly nutrients availability and stress [9]. This is predominantly mediated through its best-studied
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down-stream effectors S6K1 (p70 ribosomal protein S6 kinase 1) and 4EBP1 (eIF4E binding protein). The serine/threonine protein kinase Akt, a downstream effector of PI3K, is a critical
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mediator of mTOR activity [10]. The prominence of Akt/mTOR integrity have been documented by several empirical evidences to promote cell survival [11, 12] and proliferation which is mainly
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mediated through S6K activation. In line with this, there are pile of concrete evidence indicating that Akt/mTOR inhibitors like Rapamycine may be regarded as therapeutic agents to impede
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cancerous proliferation of different cell types [13, 14]. Notably, ROS such as superoxide (O2−) and hydrogen peroxide (H2O2) could also regulate tyrosine phosphorylation [15, 16] and may suppress mTOR activity through Akt inhibition [17]. Nevertheless, there are also recent evidences implying ROS-induced repression of mTOR may be downstream to tuberous sclerosis complex (TSC) signaling node on the peroxisome which functions as a cellular sensor for ROS to regulate mTOR and then autophagy [18]. Taken together, Akt/mTOR signaling seems to get remarkably affected by both oxidative stress and metformin. Therefore, theoretically it could be assumed that Akt/Mtor may mediate anti-oxidant effects of the drug. Accordingly, in the present work we aimed to determine the correlation between Akt/mTOR activity and metformin induced protection against oxidative stress. Since the H2O2 induced insult was associated with dampened Akt/mTOR, the main purpose was to find whether metformin protective doses enhance Akt/mTOR activity 3
ACCEPTED MANUSCRIPT simultaneously. To this end PC12 cells as commonly used neural like cells were exposed to H2O2 in IC50 concentrations and effect of metformin pretreatment was determined at transcriptional and post-translational levels of PI3K/Akt/mTOR/S6K signaling components. The
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concurrent changes in antioxidants’ status; catalase (CAT) and superoxide dismutase (SOD)
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activity and glutathione (GSH) levels, were also evaluated in stressed cells exposed to protective concentrations of metformin that has been identified as an anti-oxidant agent [19].
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2. Experimental Procedures 2.1. Cell culture and treatment
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PC12 cells (National cell bank, Pasteur institute of Iran) were maintained in high glucose (4500 mg/L) DMEM containing heat inactivated fetal bovine serum (FBS) (10% v/v), Penicillin
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(100 units/ml) and Streptomycin (100 µg/ml) (all from Invitrogen) at 37 °C in 95% air and 5% carbon dioxide. PC12 cells, at passage number 8-10, were seeded in 96-well plates (JET BioFil)
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at a density of 1×104 cells per well in serum free DMEM medium (Invitrogen) and were allowed to attach and expand for 24 h. H2O2 at the concentration of 150 µM was used to induce oxidative
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stress in PC12 cells. Pre-treatment with varying concentrations of freshly prepared Metformin (0.1, 0.5, 1, 3, 5, 7 mM) solution was performed for 60 min prior to stress induction by H2O2.
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Cells were then subjected to experimental analyses following H2O2 exposure for 24 h. A negative control cell group containing culture medium alone was also included as reference cells.
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2.3. Cell survival assay
Cell viability was grossly assessed by the stress related changes as determined in three separated fiedls in each photomicrographs. The mitochondrial integrity was also evaluated by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction to purple formazan [20]. Briefly, cells were incubated with MTT solution (final concentration 0.1-7 mg/mL) for 3 h at 37 °C. The medium was carefully removed by aspiration and formazan crystals were dissolved in 100 µl of dimethyl sulfoxide (DMSO). The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm and was reported as the percentage of cell viability compared to control.
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ACCEPTED MANUSCRIPT 2.4. Superoxide dismutase activity assay Superoxide dismutase (SOD) activity was analyzed by the methods of Kakkar et al [21]. Briefly the assay mixture containing appropriate amounts of nicotinamide adenine dinucleotide,
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phenazine methosulphate and nitroblue tetrazolium (750, 186 and 300 µM respectively) were
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subjected to react with SOD present in cellular lysate. Following adequate incubation in 30 οC (90 sec), the mixture was stirred after n-butanol addition. The measured absorbance at 560 nm
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was considered as the color intensity of amino blue tetrazolium formazan (ABTF), the chromogen in butanol. Samples which resulted in 50% inhibition of total nitroblue tetrazolium
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reduction to ABTF per each mg of protein, was considered to possess one unit of enzymatic activity.
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2.5. Catalase activity assay
Catalase (CAT) activity was determined by the method of Aebi [22]. Simply cell lysate was
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mixed with phosphate buffer (50mM, pH 7.0) and subjected to react with the freshly prepared H2O2 (30mM). The rate of decomposition of H2O2 measured by the mixture’s absorbance at
2.6. GSH level analysis
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240 nm was considered as the CAT activity.
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Total GSH levels were measured in whole cell lysates according to the rate of colorimetric change of 5,5′-dithiobis-2-nitrobenzoic acid (Ellman’s reagent) as determined by the
µmol/mg.
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spectrophotometric method and absorbance at 405 nm [23]. GSH concentration was expressed as
2.7. Immunoblot Analysis The cells were washed with PBS scraped in ice-cold RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% sodium deoxycholate, 1% NP-40, 0.5 mM PMSF), and incubated on ice for 30 minutes. The cellular debris was removed by centrifugation (8000g for 15 min) at 4 °C and the cell lysate was carefully transferred to the microcentrifuge tubes. Proteins in samples standardized by Bradford assay [24], were separated by SDS electrophoresis and transferred to PVDF membranes. To estimate the amount of target proteins, membranes were then incubated with specific primary antibodies against Akt1/2/3/3 (H-136) and p-Akt1/2/3 (Ser 473), PI3 Kinase Class III, phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199), mTOR, phospho5
ACCEPTED MANUSCRIPT mTOR (Ser248), p70S6 Kinase and phospho-p70 S6 Kinase (Thr389). Antibodies were obtained from Cell Signaling Technology and prepared in 1:1000 v/v dilutions except for Akt1 and p-Akt1 which were purchased from Santa Cruz and used in 1/600 v/v dilution. To visualize protein
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bands, the blots were incubated with horseradish peroxidase-linked secondary antibody (#7074)
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(1:10000 v/v; Cell Signaling Technology) which is directly detectable by chemiluminescence kit reagent (Amersham). The attained scans were then analyzed semi-quantitatively using Image J
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software in proportion to β-actin band intensity as internal control. 2.8. Quantitative Real-Time PCR
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Total RNA was extracted from cultured cells using NucleoSpin II kit (Macherey-Nagel GmbH & Co KG) and cDNA was synthesized using Revert Aid first strand cDNA synthesis kit
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(Thermo Fisher Scientific Inc.), all according to the manufacturers’ instructions. Quantitative RTPCR was performed on an ABI 7500 real time PCR system (PE Applied Biosystems, Foster City, CA, USA) using Maxima SYBR Green/ROX qPCR Master Mix according to the manufacturer’s
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recommendations with primer pairs listed in Table 1. β-actin was used as the reference housekeeping gene. The relative expression of mRNA transcripts, standard error means and p-
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values for differences in fold-expression were determined by the relative expression ratio method
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via the Relative Expression Software Tool-Multiple Condition Solver (REST-MCS) [25]. 2.9. Statistical Analysis
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All results represent the mean value from at least three separate experiments each with at least two replicates. All the data with the exception of those obtained at real-time PCR, were statistically analyzed using SPSS software. Normality of data was verified using the ShapiroWilk test. Analysis was performed with one-way ANOVA followed by Tukey post-hoc test and a P-value < 0.05 was considered as significant. Data are presented as mean ±SEM with the exception of relative fold changes in gene expression data which are presented as mean ±SD.
3. Results
3.1. Metformin does not exert dose-dependent protection against H2O2-induced injury in PC-12 cell
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ACCEPTED MANUSCRIPT According to the presumptive diverse roles of metformin as a pro-survival [1] or proapoptotic [26] agent and for the differences in the experimental setup (eg. different cell lines) a dose response assay was primarily performed to determine the protective metformin
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concentrations. Photomicrographs show that the presence of metformin together with H2O2
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attenuates morphological alterations caused by H2O2 as defined by the ratio of stressed round shaped cells (Fig. 1a,b). Nevertheless, such impact was not detectable in metformin
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concentrations higher than 0.5 mM. Similarly, metformin (at 5 discreet concentrations ranging from 0.1 to 5 mM) did not show consistent dose-dependent responses in MTT assay. That is
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while incubation of the PC12 cells with metformin at 1, 3, and 5 mM concentrations did not result in restoring cell viability, metformin exposure at 0.1 and 0.5 mM concentrations rendered
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PC12 cells nearly resistant to H2O2 (Fig. 1c). Given metformin alone (at 5 discrete concentrations ranging from 0.1 to 5 mM) did not change MTT assay results (data not shown), such alterations are not seemingly due to metformin impact on intact cells. H2O2 concentration
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used to cause oxidative stress (150 µM) was selected according to the previously reported H2O2
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LD50% of 150 µM in a 24 h period of exposure as identified by our previous data [27].
3.2. The H2O2 induced decline in CAT and GSH was reversed by metformin
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To evaluate cells’ antioxidant strength, metformin effects was assessed on SOD, CAT and GSH as major detoxifying agents which degrade superoxide and H2O2 [28, 29]. According to the
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obtained data the antioxidant activity of SOD and CAT and the content of GSH decreased by H2O2 (150 µM) treatment in PC12 cells with higher confidence interval for CAT. Metformin treatment was not associated with any change in H2O2 induced escalation in SOD activity. Instead, as obtained by CAT and GSH assessment, PC12 cells exhibited improved CAT and GSH while pretreated with metformin low concentrations. Noteworthy, the improving effect of metformin on CAT and GSH vanished in doses higher than 1 mM and 0.5 mM respectively (data not shown). This may be corroborated with our MTT data in which metformin could not preserve cells’ viability while applied in high concentrations. Thus in order to evaluate the molecular alterations concurrent with metformin alleviating effects we proceeded to next experiments with metformin at 0.1, 0.5 and 1mM dosages (Fig 2).
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ACCEPTED MANUSCRIPT 3.3. Protective effects of metformin concurred with enhanced PI3K/Akt/mTOR/S6K activity Impact of oxidative stress on PI3K/Akt/mTOR/S6K may be extensively context sensitive
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[17]. Several concrete evidences suggest ROS may suppress PI3K/S6K while others imply in
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pro-survival concentrations, ROS might activate PI3K/S6K ROS [30, 31]. According to our data, in our set of experiments H2O2 effectively inhibited PI3K/S6K activation as determined by the
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proportion of phosphorylated to un-phosphorylated PI3K/S6K constituents and the corresponding down streams mTOR and S6K. The relatively coordinated changes in PI3K, Akt, mTOR and
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S6K phosphorylation was suggestive of profound impression by H202. Notably, metformin augmented PI3K/S6K activity in the low dosages (0.1 and 0.5 mM) while in 1 mM it left the
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H2O2 induced PI3K/S6K repression almost entirely unaffected or even slightly intensified. As presented in figure 3, nearly all corresponding changes due to 1 mM metformin share the property of showing significant decline compared to control while it is not the case for the lower
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concentrations (0.1 and 0.5 mM). Taken together with the MTT data, this may imply the remarkable pro-survival effect of metformin was almost coincident with the restoring PI3K/S6K
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pathway.
proteins activation
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3.4. Changes in PI3K/Akt/mTOR/S6K mRNA transcripts inversely correlate with that of
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Quantitative real time PCR results of three independent experiments showed there is a significant shift towards enhanced transcription of PI3K/Akt/mTOR/S6K cascade proteins with increasing dosages of metformin (0.1 to 1 mM). As shown in figure 4, seemingly the upward change of transcripts in 1 mM concentration of metformin is the only case of over-expression that was strong enough to markedly surpass the corresponding one in control that is determined by 1 in y axis. The relevant refinements were also performed in the legend and also the pertinent result section.The maximal mRNA transcript was detected in 1 mM which surpassed that of control
cells. Interestingly as a common future, metformin in all included doses (0.1, 0.5 and 1 mM) seemingly reversed the proportion of Akt and mTOR transcripts to the up-stream effector PI3K. Nevertheless, here we could not suggest any correlation with protective effects of metformin since the mRNA levels did not consistently exceed that of control group in both protective concentrations of metformin (i.e.; 0.1 and 0.5 Mm) (Fig4).
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ACCEPTED MANUSCRIPT 4. Discussion
Metformin antioxidant properties have been shown to reserve endothelial function [32] and
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protect against cardiovascular [33] and central [34] complications in diabetic subjects, however;
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no evidence has been reported considering PI3K/S6K implication. Results of the present work demonstrate that PI3K/S6K activity is restored during metformin induced protection. In this
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connection, our data suggest metformin may enhance cell survival in the face of oxidative stress at least partly through strengthening antioxidant systems particularly GSH and CAT.
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Basically, mitochondrion is the primary target of metformin within the cell in which metformin transiently inhibits complex I in electron transport chain. In the view of diabetes
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therapy, this results in a drop in energy charge [35] and consequently in AMP/ATP proportion which in turn activates AMP-activated protein kinase (AMPK) which negatively regulates mTOR/ S6K activation [36, 37]. On the other side, the same mechanism may also account for the
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hampered ROS production in reverse-electron flux at respiratory-chain complex 1 by metformin [38]. Besides reducing ROS levels, metformin administration may also end with strengthened
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antioxidant systems [39, 40] which might contribute to the documented nephro-protective effects of metformin in diabetes mellitus [41].
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According to our findings, low dose metformin was efficient enough to retrieve CAT and GSH despite lack of SOD restoration. This is somehow verified by previous works describing
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metformin may enhance CAT activity and GSH levels [42, 43, 44], while there is not enough evidence supporting SOD augmentation by metformin [45]. Given that oxidative stress regulates the activity of the cell survival factor Akt [35] we sought to correlate Akt/mTOR cascade to H2O2 toxicity. Having in mind H2O2 impact on Akt activity may strictly depend on cell type as well as exposure conditions [17], our results described a marked decrease in phosphorylation of Akt/mTOR inactive constituents in H2O2 treated PC12 cells. This is in agreement with previous studies indicating H2O2 induces a drop in Akt kinase activity in PC12 cells [46] which may hypothetically take part in the induced injury [47, 48]. As an effector downstream to Insulin receptor, PI3K may get activated upon Insulin binding. Metformin is empirically considered as an Insulin sensitizer in spite that it is devoid of metabolites or conjugates that could activate Insulin receptors [46]. This is noteworthy that Insulin sensitizing property of metformin has been supported by in vitro [50] and in vivo [51] 9
ACCEPTED MANUSCRIPT evidences implying metformin enhances insulin function by improving Insulin receptors-linked tyrosine phosphorylation. In line with this, low dose metformin has been shown to affect neural injury induced by glucose-oxygen deprivation through modulating phosphorylation of β-subunit
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tyrosine in Insulin receptor [52].
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Our data also described a transient PI3K/S6K signaling activation in the low dose (0.1 or 0.5 mM) while it was coincident with significant protection. This primary PI3K/S6K augmentation
Akt/mTOR activation by low dose metformin [52].
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may be the result of neutralizing H2O2 effects [46] or more probably, the direct consequent of
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Unexpectedly, our data shows the slightly higher dose of metformin (1 mM) did not keep on reversing PI3K/S6K activity against H2O2 toxicity. Actually, in some cases PI3K/S6K
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suppression even enhanced in this concentration. Such intensified PI3K/S6K suppression might be ascribed to switching between downstream effectors. That is based on empirical evidences, metformin may reasonably suppress PI3K/S6K activity by AMPK stimulation following
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interfering with the mitochondrial electron transport as described before. This way, activated AMPK switches cells from an anabolic to a catabolic state, thus shutting down the ATP-
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consuming proliferative pathways like mTOR/S6K signaling [53]. The observed S6K suppression by metformin increasing doses may indicate that such interference with Akt/S6K
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signaling may commence even in very low concentrations of the drug. Noteworthy, AMPK also has been shown to exert pro-survival effects in various cell types
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[54, 55], but here we can’t add anything about AMPK involvement as we did not examine AMPK activity. However, this presumptive AMPK stimulation by metformin might explain the opposite changes observed in PI3K/S6K by metformin at relatively higher concentration of 1 Mm. Intriguingly, in contrary to post translational effects of metformin, our results on mRNA assay identified that the transcriptional levels of PI3K/S6K signaling molecules were enhanced by any enhancement in metformin exposure. There are concrete evidences implying downstream components like mTOR may negatively control the activity of Akt/mTOR signaling [56, 57]. In this view, the initially inversed PI3K/S6K transcription at metformin low concentrations may be accountable for the transient augmentation of Akt/mTOR signaling by the low concentration of metformin. Nonetheless the reverse correlation between transcriptional and translational levels of
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ACCEPTED MANUSCRIPT PI3K/S6K constituents might be suggestive of a negative feedback loop, we did not find any
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evidence implying relevant gene transcripts are regulated by activated Akt or PI3K.
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5. Conclusion
Collectively, the present work shows metformin induced protection in PC12 cells is associated with Akt/mTOR restoration however using kinase inhibitors would substantially
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contribute to verify Akt/mTOR implication. While the absence of pro-survival effect of metformin was coincident with dismissing GSH elevation, it needs to be unraveled whether these
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anti-oxidants properties is a direct consequent of PI3K/S6K activity. Lastly regarding probable differences between the relevant intracellular cascades, examining alternative pathways and in
neuroprotective effects of the drug.
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Acknowledgements
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particular considering virtual neural cultures, may shed may shed light on literally
This study was supported financially by Neuroscience Research Center, Shahid Beheshti
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University of Medical Sciences. The authors thank Dr. Khodagholi F for valuable guidance on
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presentation.
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PC12 experiments and Dr. Talebi for effective discussion on the real time PCR results
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Figure legends Fig. 1. Effect of metformin on H2O2 induced cell injury as determined by MTT assay and morphological alterations. A 24 h period of H2o2 (150 µM) exposure caused a significant decline in PC12 cells viability. The representative photomicrographs of PC12 cells (a) demonstrate the corresponding substantial decline in round forms (b) due to protection induced by metformin at 0.1 and 0.5 mM concentrations. In the same concentrations MTT assay data (b) agrees the antioxidant activity of metformin. Values are expressed as mean ± SEM from three independent experiments, each of which including four replicates. *p < 0.05, **p < 0.01, ***p < 0.001 versus control; #p
