Resveratrol promotes cellular glucose utilization in primary cultured cortical neurons via calcium-dependent signaling pathway

Available online at www.sciencedirect.com

Journal of Nutritional Biochemistry 24 (2013) 629 – 637

Resveratrol promotes cellular glucose utilization in primary cultured cortical neurons via calcium-dependent signaling pathway Jun-Qi Zhang a, 1 , Peng-Fei Wu a, 1, Li-Hong Long a, b, c, 1 , Yu Chen a , Zhuang-Li Hu a, b, c , Lan Ni a, b, c , Fang Wang a, b, c , Jian-Guo Chen a, b, c,⁎ a

Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China b Key Laboratory of Neurological Diseases (HUST), Ministry of Education of China, Wuhan, Hubei 430030, China c The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China

Received 19 July 2011; received in revised form 19 February 2012; accepted 28 February 2012

Abstract Background and Purpose: Impairment of glucose utilization contributes to neuronal degeneration of Alzheimer's disease patients. Cellular glucose utilization can be regulated by calcium-dependent signaling pathways. Resveratrol (RSV) is a plant-derived polyphenol with multiple beneficial effects, including neuroprotection and metabolic improvement. Here, we investigated the effect of RSV on neuronal calcium signal and glucose utilization. Experimental Methods: Primary culture of cortical neurons, calcium imaging, 2-NBDG assay and western blotting were employed to investigate RSV-mediated effects on neuronal calcium signal and glucose utilization. Results: RSV elevated intracellular calcium in cortical neurons via modulation of secondary messenger system including nitrous oxide, cGMP and cAMP. Secondarily, a calcium-dependent enhancement of neuronal glucose utilization after RSV treatment was observed. The effects on neuronal glucose utilization are largely dependent on RSV-induced calcium-dependent AMP-activated protein kinase activation. Conclusion: Our findings show that activation of calcium-dependent signaling pathways by RSV may convey improvements of neuronal glucose utilization. © 2013 Elsevier Inc. All rights reserved. Keywords: Resveratrol (RSV); Calcium; Glucose utilization; AMP-activated protein kinase

1. Introduction Neurons are particularly sensitive to fluctuations in energy levels. Energy deficiency is tightly related to neuronal survival and viability and also contributes to age-related disorders, e.g., Alzheimer's disease (AD) [1,2]. Glucose utilization provides energy for functional neuronal activities. A family of glucose transporters (GluTs) is believed to be responsible for the majority of glucose utilization in brain [3,4]. Inhibition of neuronal glucose utilization may decrease neuronal activity and precede neuronal degeneration. Restrictions to glucose availability increase the sensitivity of primary neurons to glutamate excitation [5,6]. The impairment of neuronal glucose utilization in the brain of AD patients may precede neuronal degeneration [7,8]. Previous studies support the view that nutrients can serve as trophic factors to promote neuronal repair and survival following injury [9]. Thus, nutrients may improve the neurological disorders including AD. ⁎ Corresponding author. Department of Pharmacology, Tongji Medical College, HUST, Wuhan, Hubei 430030, China. Tel.: + 86 27 83692636; fax: + 86 27 83692608. E-mail addresses: [email protected], [email protected] (J.-G. Chen). 1 Equally contributed authors. 0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2012.02.015

Resveratrol (RSV), a polyphenol widely found in red wine, peanuts and pomegranate, has received tremendous attention over the past couple of decades, due to its benefits such as antioxidation, regulation of energy metabolism, neuroprotection and anti-aging effect [10–13]. Calorie restriction (CR) exhibits anti-aging effects on many species via metabolic improvements and RSV exerts multiple beneficial effects similar to those associated with CR [14–20]. Thus, more evidences support the notion that a number of benefits of RSV, such as antiaging and neuroprotection are attributable to the multiple metabolic improvements [20,21]. Recently, it has been reported that RSV inhibits Aβ accumulation via calcium-dependent signaling pathway [21]. Calcium is an important second messenger in cellular energy metabolism, involved in the docking and fusion of glucose transporter 4 (GluT4) vesicles with plasma membrane [22,23]. AMP-activated protein kinase (AMPK), a critical regulator of cellular energy metabolism, is also indirectly activated by the activation of Ca2 + sensitive calmodulin-dependent protein kinase kinases (CaMKKs) [24,25]. In particular, AMPK activation results in the increase in cellular glucose uptake [26,27], suggesting that intracellular calcium signal may regulate neuronal glucose utilization by AMPK activation. Furthermore, a transient elevation of intracellular calcium concentration promotes the vesicle exocytosis and translocation of GluT3. This exocytosis can be inhibited by amyloid beta-peptide [28,29].

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However, until now, it is not fully understood the exact mechanism by which RSV regulates cytosolic calcium levels. Therefore the precise mechanism underlying RSV -mediated calcium signaling was explored in the present study. Moreover, since previous studies show that CR can facilitate cellular glucose utilization [30,31], it is likely that RSV may produce a mimetic effect of CR on glucose utilization in neurons via calcium-dependent signaling pathways. Thus, in this study, we intended to explore the effect of RSV-triggered intracellular calcium events on neuronal glucose utilization as well as the underlying mechanisms. 2. Materials and methods 2.1. Materials Resveratrol (RSV), thapsigargin (TG), cyclopiazonic acid (CPA), BAPTA-AM, KT5823, dithiothreitol (DTT), Compound C, ICI 182780, BFA, nicotinamide, 7-oxo7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid-acetic acid (STO609), NG-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin1-one (ODQ), 5-aminoimidazole-4-carboxamide riboside (AICAR), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), and 9(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) were obtained from SigmaAldrich (St. Louis, MO, USA). NBDG and FM1-43 was obtained from Molecular Probes (Eugene, OR, USA). Fura-2 AM was obtained from Biotium (Hayward, CA, USA) and dissolved in dimethylsulfoxide (DMSO) and stored at − 20 °C. Modified Dulbecco's Eagle's medium (DMEM)/F12 and B27 supplement were obtained from Gibco Invitrogen Corporation (Carlsbad, CA, USA). Antibodies directed against AMPK, pAMPK were obtained from Cell Signaling Technology. Other general agents were available commercially. All the drugs were prepared as stock solutions and diluted to the final concentrations before application. The final concentration of DMSO was less than 0.1%. 2.2. Cell culture Primary culture of cortical neurons was prepared as described in our previous studies [32]. All experimental procedures were approved by the University of Animal Welfare Committee of Huazhong University of Science & Technology. Briefly, the cortex of neonatal Sprague–Dawley rats (Day 0–2) obtained from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science & Technology, were dissected and rinsed in ice-cold Dulbecco's phosphate-buffered saline. The dissected tissues were treated with 0.125% trypsin in Hanks' balanced salt solution for 25 min at 37°C and mechanically dissociated using a fire-polished Pasteur pipettes. Cells were collected by centrifugation and resuspended in DMEM/ F12 with 10% fetal bovine serum. The contents and concentrations of DMEM/F12 were seen in Table 1. Cells (20,000–40,000) were plated on poly-D-lysine coated coverslips and kept at 37°C in 5% CO2. After 24 h, the culture medium was changed to neurobasal supplemented with 2% B27 and 2.5 mM glutamine. Astrocytes were minimized by treating the culture with cytarabine (10 μM) on Day 3. The cortical neurons were fed with fresh medium every 2 days. Experiments were performed on Day 7–9. 2.3. Measurement of intracellular free calcium Digital calcium imaging was performed as described by Ming et al [33,34] with some modifications. For [Ca2 +]i measurements, cortical neurons were washed three times with artificial cerebrospinal fluid (ACSF) containing (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.3). Then, cells were incubated with 1 μM fura-2 AM for 30 min at 37°C and subsequently washed three times with ACSF to remove the excess extracellular fura-2/AM. Coverslips were then mounted on a chamber positioned on the movable stage of an inverted microscope (TE2000, Nikon, Japan), which is equipped with a calcium imaging system (PTI, Birmingham, UK). The cells were superfused by ACSF at a rate of 2 ml/min for 10 min. Fluorescence was excited at wavelengths of 340 nm for 150 ms and 380 nm for 50 ms at 1 s interval by a monochromator (PTI K-178-S) and the emitted light was imaged at 510 nm with a video camera (CoolSNAP HQ2; Roper, Trenton, NJ, USA) through fluor oil-immersion lens (Nikon) and a wide band emission filter. F340/F380 fluorescence ratio was recorded and analyzed by MetaFluor version 6.3 software, which was used as an indicator of [Ca2 +]i independent of intracellular fura-2 concentration. All experiments were repeated at least three times using different batches of cells. 2.4. Glucose uptake assay using 2-NBDG Cellular glucose utilization was measured using NBDG assay as described by Lloyd et al [35,36] with some modifications. Neurons were pretreated with RSV (10 μM) or vehicle (DMSO) for 20 min. Then neurons were washed twice and incubated in Earle's solution containing: (in mM) 116 NaCl, 5.4 KCl, 0.8 MgSO4, 1.0 NaH2PO4, 1.8 CaCl2, and

Table 1 The contents and concentrations of DMEM/F12 medium Components

mM

Glycine L-Alanine L-Arginine hydrochloride L-Asparagine-H2O L-Aspartic acid L-Cysteine hydrochloride-H2O L-Cystine·2HCl L-Glutamic Acid L-Glutamine L-Histidine hydrochloride-H2O L-Isoleucine L-Leucine L-Lysine hydrochloride L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine disodium salt dihydrate L-Valine Biotin Choline chloride D-Calcium pantothenate Folic acid Niacinamide Pyridoxine hydrochloride Riboflavin Thiamine hydrochloride Vitamin B12 i-Inositol Calcium chloride (CaCl2) ( anhydrous ) Cupric sulfate (CuSO4-5H2O) Ferric nitrate (Fe(NO3)3·9H2O) Ferric sulfate (FeSO4·7H2O) Magnesium chloride (anhydrous) Magnesium sulfate (MgSO4) (anhydrous) Potassium chloride (KCl) Sodium bicarbonate (NaHCO3) Sodium chloride (NaCl) Sodium phosphate dibasic (Na2HPO4) anhydrous Sodium phosphate monobasic (NaH2PO4·H2O) Zinc sulfate (ZnSO4·7H2O) D-Glucose (dextrose) Hypoxanthine Na Linoleic acid Lipoic acid Putrescine·2HCl Sodium pyruvate Thymidine

0.25 0.05 0.699 0.05 0.05 0.0998 0.1 0.05 2.5 0.15 0.416 0.451 0.499 0.116 0.215 0.15 0.25 0.449 0.0442 0.214 0.452 0.0000143 0.0641 0.0047 0.00601 0.0166 0.00971 0.000582 0.00644 0.000502 0.07 1.05 0.0000052 0.000124 0.0015 0.301 0.407 4.16 29.02 120.61 0.5 0.453 0.0015 17.51 0.015 0.00015 0.00051 0.000503 0.5 0.00151

26 NaHCO3 (pH 7.3). At each culture well, 2-NBDG, a fluorescein derivative of D-glucose, was added at a final concentration of 300 μM and the neurons were incubated with 2-NBDG for 30 min. Then the neurons were superfused with Earle's solution for 5 min to clear 2-NBDG from the bath and eliminate background fluorescence. Following washout, an image of neuron were acquired using Zeiss LSM510 confocal microscope (Zeiss, Jena, Thuringia, Germany) with band-pass filters for excitation (peak 485 nm) and emission (peak 505 nm). More than 10 cells were selected for the determination of 2-NBDG fluorescence. Average fluorescence intensity (Iavg) of the cells was determined with ImagePro Plus software. 2.5. Western blotting After treatment, cortical neurons were washed twice with ice-cold PBS and then lysed on ice in extraction buffer containing 50 mM Tris-base (pH 7.4), 100 mM NaCl, 1% NP-40, 10 mM EDTA, 20 mM NaF, 1 mM PMSF, 3 mM Na3VO4, and protease inhibitors. The homogenates were centrifuged at 12,000 × g for 10 min at 4°C. Supernatant was separated and stored at − 80°C until use. Protein concentration was determined by using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Protein samples (20 μg) were separated by 10% SDS-polyacrylamide gel and then transferred to nitrocellulose membranes. After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, transferred membranes were incubated overnight at 4°C with different primary antibodies (AMPK: 1:500 dilution; β-actin: 1:400 dilution). Following three washes with TBST, membranes were

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then incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000) in TBST with 3% nonfat milk for 1 h at room temperature. After repeated washes, membranes were reacted with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for 5 min, and visualized with X-ray films (Kodak X-Omat, Rochester, NY, USA). The films were scanned and the optical density of the bands was determined using Optiquant software (Packard Instrument). Normalization of results was ensured by running parallel western blots with β-actin.

2.6. Statistical analysis The amplitude of [Ca2 +]i transient represents the difference between base line concentration and the transient peak response to the RSV stimulation. The amplitude of [Ca2 +]i elevation upon the basal [Ca2 +]i was normalized to the basal [Ca2 +]i, which is taken as 100%. Data are presented as means±S.E.M. and analyzed using SPSS 11.0 software. Comparison amplitude of [Ca2 +]i after different treatments on the same cell was evaluated using the paired Student's t test. Multiple comparisons were tested by one-way analysis of variance followed by Duncan's multiple-range test. Differences were considered significant at Pb.05.

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3. Results 3.1. RSV increase [Ca2 +]i in a dose-dependent manner by mobilization of intracellular calcium stores In the first set of experiments, we employed the calcium imaging technique to investigate the dynamic alteration of intracellular calcium mediated by RSV in cultured rat prefrontal cortical neurons. The vehicle, 0.1% DMSO, exhibited no effect on the [Ca2 +]i of cortical neurons (data not shown). Incubation of cortical neurons with RSV (5–100 μM) increased [Ca2 +]i in a dose-dependent manner by 42.30±0.53% over basal levels at 5 μM, 138±1 % at 10 μM, 201±8 % at 50 μM and 365±5% at 100 μM (n=12, Fig. 1A–B). Thus, the concentration of 10 μM was selected for further experiments. A significant elevation in [Ca2 +]i induced by RSV (10 μM) was observed within 30 s following application of RSV. In the absence of extracellular Ca2 +, 10 μM

Fig.1. RSV produces a dose-dependent [Ca2 +]i increase by mobilization of intracellular calcium stores. (A) [Ca2 +]i responses to repeated 10 μM RSV stimulation under control condition in primary cultured cortical neurons. The time intervals between the stimuli were 10 min. (B) Summary of the maximal response in [Ca2 +]i for different concentrations of RSV (n= 12). (C) The superfusion of cultured neurons was switched from Ca2 +-containing solution to Ca2 +-free solution when 10 μM RSV was added. (D) Summary data showing RSV-evoked [Ca2 +]i increase in both Ca2 +-containing and Ca2 +-free solution (n=12). (E) Pre-incubation with CPA and TG, prevented RSV-evoked [Ca2 +]i increase. (F) Quantitative analysis of Ca2 + release from intracellular stores, indicating that intracellular calcium store is necessary and sufficient for RSV-induced [Ca2 +]i rise (n= 12). ##Pb.01 compared to control; **Pb.01 compared to RSV.

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RSV also induced a significant increase in [Ca2 +]i (137±3 % over basal levels, n=15, Fig. 1C–D), with no significant difference from that in the presence of extracellular Ca2 +. Furthermore, preincubation with 10 μM CPA and 10 μM TG, two inhibitors of sarcoplasmic/endoplasmic reticulum Ca2 +-ATPase to deplete the

intracellular calcium store in neurons bathed in Ca2 +-containing medium, RSV almost failed to evoke a significant increase in [Ca2 +]i (n=12, Fig. 1E–F). These results indicate that Ca2 + release from intracellular stores is necessary and sufficient for RSV-induced [Ca2 +]i rise.

Fig.2. Activation of ER-NOS-GC-PKG signaling pathway contributes to the regulatory effect of RSV on [Ca2 +]i. (A) Preincubation with estrogen receptors inhibitor ICI182780 exhibited inhibitory effect on calcium-rising effect of RSV. (B) Quantitative analysis of the effect of ICI182780 on calcium-rising effect of RSV (n= 10). (C) Pre-incubation with NOS inhibitor LNAME partially abolished calcium-rising effect of RSV. (D) Quantitative analysis of the effect of NOS inhibition on the calcium-rising effect of RSV (n= 16). (E) Pre-incubation with GC inhibitor ODQ partially abolished calcium-rising effect of RSV. (F) Quantitative analysis of the effect of GC inhibition on the calcium-rising effect of RSV (n= 16). (G) Pre-incubation with PKG inhibitor KT5822 partially abolished calcium-rising effect of RSV. (H) Quantitative analysis of the effect of PKG inhibition on the calcium-rising effect of RSV (n= 16). ##Pb.01 compared to control; **Pb.01 compared to RSV.

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3.2. Activation of ER-NOS-GC-PKG signal pathway contributes to the regulatory effect of RSV on [Ca2 +]i Several reports indicate a critical role of nitrous oxide (NO) synthase (NOS) activity in the biological effect of RSV [37–39] and NO-guanylyl cyclase (GC) kinase signaling pathway attributes to [Ca2 +]i rise [40,41]. RSV belongs to the type 1 class of estrogens, which can bind to estrogen receptors (ER) and produce wide biological effects, including NOS activation [42,43]. To determine whether estrogen receptor activation by RSV contributed to its calcium-rising effect, an estrogen receptor antagonist, ICI.182780 (10 μM) was incubated with cortical neurons for 10 min. It was found that bath application of ICI.182780 only partially prevented the effect of RSV on [Ca2 +]i from 174±13 % over baseline to 104±13% (n=10, Pb.01, Fig. 2A and B). Then, we asked whether the effect of RSV can be reduced by NOS inhibition. Incubation of cortical neurons with NOS inhibitor N-nitro-larginine methyl ester (L-NAME, 100 μM), partially inhibited calciumrising effect of RSV from 166±18 % to 79±11 % over basal levels (n=12, Pb.01, Fig. 2C and D), suggesting that production of the

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secondary messenger NO contributes to RSV action. The classical signaling pathway activated by NO is GC-cGMP-PKG (protein kinase G) pathway. So the effects of GC inhibitor (ODQ) and PKG inhibitor (KT5823) on RSV action were observed. After pretreatment with ODQ (20 μM) or KT5823 (10 μM), RSV (10 μM) produced less increase in [Ca2 +]i from 152±11 % to 60±14 % over basal levels (n=12, Pb.01, Fig. 2E and F) and from 177±6 % to 113±6 % (n=12, Pb.01, Fig. 2G and H), respectively. These results indicate that NO-mediated NOS activation contributes to RSV-induced calcium-rising effect. 3.3. Activation of AC-PKA signaling pathway participates in RSV-induced [Ca2 +]i elevation Since blockade of ER-NOS-GC-PKG signaling pathway only partially inhibited RSV -induced [Ca2 +]i elevation, other mechanisms may also exist. Another important secondary messenger involved in RSV action is cAMP. Some reports show that RSV activates adenylate cyclase (AC) and initiates a series of cAMP-dependent effects [44,45]. To determine whether RSV-induced [Ca 2 +]i elevation is cAMP-

Fig.3. Activation of AC-PKA signaling pathway participates in RSV-induced [Ca2 +]i elevation. (A) Preincubation with AC inhibitor SQ22536 partially abolishes calcium-rising effect of RSV. (B) Quantitative analysis of AC inhibition on the calcium-rising effect of RSV (n=16). (C) Preincubation with PKA inhibitor H89 partially abolishes calcium-rising effect of RSV. (D) Quantitative analysis of PKA inhibition on the calcium-rising effect of RSV (n= 16). (E) The RSV-induced calcium-rising effect can be nearly prevented by pretreatment with NOS inhibitor L-NAME and AC inhibitor SQ22536 together. (F) Quantitative analysis of PKA inhibition in combination of NOS inhibition on the calcium-rising effect of RSV (n= 16). ##Pb.01 compared to control; **Pb.01 compared to RSV.

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Fig.4. Sirt1 and AMPK are not involved in the regulatory effect of RSV on [Ca2 +]i. (A) Preincubation with Sirt1 inhibitor nicotinamide exhibited little effect on calcium-rising effect of RSV. (B) Quantitative analysis of the effect of nicotinamide on calcium-rising effect of RSV (n=10). (C) Preincubation with AMPK inhibitor compound C had no significant effect on RSV-induced [Ca2 +]i increase. (D) Quantitative analysis of the effect of compound C on calcium-rising effect of RSV (n= 10). ##Pb.01 compared to control; **Pb.01 compared to RSV.

dependent, the AC inhibitor SQ22536 (100 μM) was employed. It was found that pretreatment with SQ22536 for 10 min decreased RSVinduced [Ca2 +]i elevation from 154±7% to 62±4 % over baseline (n=12, Pb.01, Fig. 3A and B). Furthermore, exposure of cells to H89 (20 μM), an inhibitor of PKA (protein kinase A), for 10 min, also attenuated the effect of RSV from 173±16 % over baseline to 76±5% (n=12, Pb.01, Fig. 3C and D). These results indicate that the activation of AC-PKA signaling pathway participates in RSV-induced [Ca2 +]i increase. Importantly, pretreatment of cells with AC inhibitor SQ22536 and NOS inhibitor L-NAME almost abolished the RSVinduced calcium-rising effect from 144±7 % over baseline to 16±2% (n= 12, Pb.01, Fig. 3E and F), suggesting that modulation of intracellular secondary messenger system including NO, cGMP and cAMP, is mainly responsible for RSV-induced [Ca2 +]i increase.

3.4. Sirt1 and AMPK are not involved in the regulatory effect of RSV on [Ca2 +]i Sirt1 is an important target directly activated by RSV [46,47], and we also investigate the possible role of Sirt1 in RSV-induced [Ca2 +]i increase. However, pretreatment with Sirt1 inhibitor, nicotinamide, exhibited no effect on RSV-induced [Ca2 +]i increase (RSV: 164±5%; nicotinamide+RSV: 153±8%; n=10, Fig. 4A-B) Recent reports have revealed that RSV can activate AMPK directly [48,49] and AMPK activation may be involved in calcium-rising effect mediated by hormones such as ghrelin [50]. In order to characterize the underlying role of AMPK in RSV-induced [Ca2 +]i increase, an AMPK inhibitor compound C was incubated with cortical neurons for 20 min, but no significant effect on RSV-induced [Ca2 +]i response was observed (RSV: 190±5 %, compound C: 182±4 %; n=10, Fig. 4C and D). These results demonstrated that RSV regulates cytosolic calcium levels via direct modulation of intracellular secondary messenger system, not via activation of Sirt1 and AMPK.

3.5. RSV increases neuronal glucose uptake via a calcium-dependent signal pathway We were sparked to explore whether RSV could exhibit a mimetic effect of CR on glucose utilization in cortical neurons via a calciumdependent signal pathway. We then examined the effects of RSV treatment on neuronal glucose uptake using 2-NBDG, a fluorescein derivative of D-glucose. After treatment with RSV (10 μM) or vehicle, DMSO for 30min, neurons were washed with Earle's solution without glucose, and then NBDG was added to a final concentration of 300 μM. After 30 min of incubation, neurons were observed using a twophoton fluorescence microscopy. As shown in Fig. 5A, the intracellular accumulation of NBDG significantly increased after RSV treatment, as the average fluorescence intensity (Iavg) increasing from 2940±435 to 6790±311. The membrane permeable calcium chelator BAPTA-AM could abolish the RSV effect, suggesting that acute RSV treatment facilitates glucose utilization in cortical neurons via a calciumdependent pathway. 3.6. Calcium-dependent activation of AMPK is essential for RSV mediated reinforcement of neuronal glucose uptake RSV can activate AMPK both directly [48] and indirectly as a result of RSV-induced [Ca2 +]i elevation [21]. Considering RSV elevates [Ca2 +]i rapidly, it is possible that RSV produces a rapid activation of AMPK via a calcium dependent signal pathway. It was found that exposure of neurons to RSV (10 μM) for 10 min and 30 min activated AMPK in a time-dependent way (n= 4, Pb.05, Fig. 6A). To determine the role of intracellular calcium in this rapid AMPK activation by RSV, the cortical neurons were pre-incubated with an intracellular calcium chelator, BAPTA-AM (10 μM) and an inhibitor of CaMKKβ, STO609 (100 μM), respectively, for 15 min before RSV treatment. As shown in Fig.6 B–C, both BAPTA-AM and STO609 abolished the rapid activation of AMPK caused by RSV treatment

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Fig.5. RSV reinforces neuronal NBDG uptake via a calcium-dependent signaling pathway. (A) RSV treatment increased neuronal uptake of NBDG via a calciumdependent signaling pathway. a, cortical neurons with vehicle treatment as control; b, cortical neurons with RSV (10 μM) treatment; c, cortical neurons with BAPTA-AM preincubation and RSV (10 μM) treatment. (B) Quantitative analysis of the effect of RSV on the NBDG uptake in cortical neurons (n=6). ##Pb.01 compared to vehicle; **Pb.01 compared to RSV.

(n= 4, Pb.05, Fig. 6B–C), suggesting that the rapid [Ca2 +]i elevation mediates the rapid activation of AMPK by RSV treatment. AMPK activation facilitates cellular glucose uptake. Compound C, an AMPK inhibitor, could abolish the effect of RSV on the NBDG uptake in cortical neurons, as shown in Fig. 6D–E. These results indicate that calcium-dependent activation of AMPK is essential for RSV-mediated reinforcement of neuronal NBDG uptake. 4. Discussion In the current study, we have shown that RSV produces an intracellular calcium elevation in cortical neurons via modulation of secondary messenger system including NO, cGMP and cAMP. Once calcium enters the neuron, a calcium-dependent AMPK activation is initiated. As a result, the intracellular glucose utilization is enhanced in a calcium-dependent way (Fig. 7). Resveratrol, a polyphenol that is present at high levels in the skin of grapes, nuts and red wine, attracts more and more attention for its promising and various biological effects, including antioxidation, neuroprotection and anti-aging effect. Recent study suggests an acute modulation on [Ca2 +]i by RSV [21], but the exact mechanism is far from clear. The cellular responses to RSV include regulation of gene transcription via increasing activities of Sirt1 [46,47] and estrogen receptors [42,43], modulation of intracellular secondary messenger system such as NO, cGMP and cAMP [37–39,44,45], and mimic of metabolic changes caused by

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CR [18,19]. Among the three responses of RSV, both gene transcription and metabolic changes need a relative long period. In our study, the increase in cytosolic calcium levels by RSV is rapid. Therefore, modulation of intracellular secondary messenger system seems to be the most reasonable mechanism for this acute effect. This hypothesis is supported by our observations that blocking of Sirt1 and AMPK has no influence on its rapid calcium rising effect. RSV mobilizes Ca2 + release from intracellular stores in a dosedependent way and its effect is nearly abolished by co-inhibition of ER-NOS-GC-PKG and AC-PKA signal pathways. A calcium-dependent AMPK activation can be secondarily initiated and leads to metabolic changes including the increase of cellular glucose uptake. AMPK activation also initiates regulation of gene transcription via modulation of activity of Sirt1 and FOXO3 [51,52]. Our results highlight that the regulatory effects of RSV on intracellular secondary messenger system may confer a critical role in the pharmacological action of RSV. Recent studies reveal an important role of Ca2 + in cellular energy utilization, especially glucose uptake. Calcium signal seems to improve insulin-stimulated GluT4 translocation by promoting the docking and fusion of the vesicles with the plasma membrane. Increased Ca2 + influx results in an increase in insulin-mediated glucose uptake both in normal and insulin-resistant skeletal muscle. This calcium-dependent regulator of energy utilization is CaMKKs, which contributes to the upregulation of cellular glucose utilization by indirect activation of AMPK [24,25]. In this study, a rapid calcium-dependent AMPK activation has been observed after RSV treatment. RSV produced a calcium-dependent reinforcement of glucose uptake in cortical neurons. This effect can be inhibited by inhibition of AMPK using compound C. These results suggest that nutritional agents that potentiate cellular calcium mobilization may be used to treat energy metabolism deficiencies and insulinresistant conditions. Restricting calories to 30–50% below ad libitum levels exhibits anti-aging effect and prevents age-related diseases in numerous species [14,15]. In recent years, CR mimetics has emerged as an anti-aging strategy without actually restricting food intake [16,17]. Previous studies have demonstrated that CR can inhibit nonessential energy expenditure and facilitate glucose utilization via increase in GluT4 function and membrane translocation [30,31]. The impairment of neuronal glucose utilization is important in neuronal degenerative diseases, including AD, which is referred to as type 3 diabetes [53]. Thus, CR mimetic nutritional agents, such as RSV, may serve as a novel strategy in the treatment of age-related neurological diseases. Although persistent calcium overload contributes to neuronal death in many pathological conditions, a transient intracellular calcium signal may be significantly protective via initiation of various cellular responses. For example, in our previous study, we have demonstrated that SKF83959, a selective PI-linked D1-like receptor agonist, produces a mobilization of intracellular calcium store in hippocampal neurons and astrocytes[33,54]. Recent study has revealed that SKF83959 increases FGF-2 expression in astrocytes via IP3-dependent Ca 2 + signaling and reverses l-methyl-4-phenyll,2,3,6-tetrahydropypridine-induced neurotoxicity [55]. Our present study has revealed that RSV facilitates cellular glucose uptake by mobilizing intracellular calcium stores. Thus, nutritional agents that can affect calcium signal pathways may have various biological functions and potential use in clinical medication. In conclusion, our results raise the possibility that modulation of calcium-dependent signal pathways by using nutritional approaches such as RSV preconditioning may ameliorate glucose utilization in neurons and provide a potential strategy for prevention of neuronal degenerative diseases in the future.

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Fig.6. Calcium-dependent activation of AMPK is essential for RSV-mediated reinforcement of neuronal NBDG uptake. (A) RSV (10 μM) activates AMPK rapidly in cultured rat cortical neurons. (B) Rapid activation of AMPK by RSV was inhibited by BAPTA-AM, indicating that this effect was calcium-dependent. (C) Quantitative analysis of the role of calcium involved in RSV-mediated AMPK activation. (D) Blockade of AMPK activation abolished RSV-mediated reinforcement of neuronal NBDG uptake. (F) Quantitative analysis of RSV action on NBDG uptake with or without AMPK inhibition (n= 6). ##Pb.01 compared to vehicle; **Pb.01 compared to RSV.

Fig.7. Mechanisms involved in RSV-mediated enhancement of glucose uptake in cortical neurons.

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