Cilostazol Enhances Oxidative Glucose Metabolism in Both Neurons and Astroglia without Increasing Ros Production 316 cause oxygen consumption in the brain

Cilostazol, a potent inhibitor of type 3 phosphodiesterase (PDE3), has recently been reported to exert neuroprotective effects during acute cerebral ischemic injury. These effects are, at least in part, mediated by the inhibition of oxidative cell death. However, the effects of cilostazol on glucose metabolism in brain cells have not been determined. In the present study, we examined the effects of cilostazol on the oxidative metabolism of glucose and the resultant formation of reactive oxygen species (ROS) in cultured neurons and astroglia. Cultures of neurons or astroglia were prepared from Sprague-Dawley rats. The cells were treated with cilostazol (0 30 μM) for 48 hours prior to the assay. L-[U-C]lactate ([C]lactate) or [1-C]pyruvate ([C]pyruvate) oxidation was measured. ROS production was determined using an H2DCFDA assay with a microplate reader. Forty-eight hours of exposure to cilostazol resulted in dose-dependent increases in [C]lactate and [C]pyruvate oxidation in both the neurons and astroglia. Dibutyryl cyclic AMP (0 0.5 mM) also increased [C]lactate oxidation, indicating cAMP-mediated PDH activation. In contrast, free radical formation was not affected by cilostazol in either the neurons or astroglia. Cilostazol enhanced the oxidative metabolism of glucose in both neurons and astroglia, while it did not augment ROS production.


Introduction
Brain function is completely dependent on the oxidative metabolism of glucose for ATP synthesis.The functional activation of local brain lesions triggers ATP consumption by Na + , K + -ATPase, which restores the trans-membrane Na + and K + gradients in the local cells [1].In contrast to the brain, the heart muscle is dependent on fatty acid oxidation in mitochondria for its energy production [2].A type 3 phosphodiesterase (PDE3) inhibitor has been known to enhance cardiac contractile force by enhancing fatty acid metabolism [3].Accordingly, an in vitro experiment has shown that two PDE inhibitors, enoximone and milrinone, suppress glucose oxidation in isolated cardiac myocytes [4] in accordance with Randle's hypothesis [5], implying a reciprocal control of glucose oxidation and fatty acid oxidation in living organisms.Importantly, as fatty acid is not an energy substrate for brain tissue under normal physiological conditions, the effect of PDE inhibitors on fatty acids does not affect brain energy metabolism, and the effect of PDE inhibi-tors on glucose metabolism in neural tissue has not been previously studied, as far as we know.
Cilostazol, a potent PDE3 inhibitor [6], has been used for effective secondary prevention of ischemic stroke for the past several years [7][8][9].It exerts a potent antithrombotic action through the inhibition of PDE3 in both platelets and vascular endothelial cells.Unfortunately, however, the actions of cilostazol on brain parenchymal cells (i.e., neurons and glial cells) have not been examined extensively.Cilostazol also plays a neuroprotective role during acute [10] and subacute [11] phases of cerebral ischemia.This action is thought to depend on increases in the cyclic adenosine 3',5'-monophosphate (cAMP) concentration by inhibiting PDE, which, in turn, activates cAMP-dependent protein kinase (PKA) and the cAMP-responsive-element-binding protein (CREB) phosphorylation pathway, resulting in anti-apoptotic effects [12,13].Moreover, cilostazol is reported to scavenge reactive oxygen species (ROS) [10].In brain tissue, mitochondria are believed to be a main source of ROS be-cause oxygen consumption in the brain is high and ROS formation is further enhanced during ischemia, leading to cell death [15].
The goal of the present study was to investigate the effect of cilostazol on glucose metabolism in cultured neurons and astroglia.We also examined the effect of cilostazol on ROS production in these two types of cells.

Animals
Timed-pregnant Sprague-Dawley rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan).All animal procedures were performed in accordance with The Animal Experimentation Guidelines of Keio University School of Medicine and were approved by the Laboratory Animal Care and Use Committee of Keio University.

Preparation of Cells
Primary astroglial cultures were prepared from the cerebral cortex of rat pups 24 to 48 hours after birth [16].Approximately on day 10, the adherent cells were treated with trypsin-EDTA solution, suspended in fresh highglucose medium (diluted 1:4), and placed in uncoated 24-well culture plates (Sumitomo Bakelite, Tokyo, Japan), 12-well culture plates (Nalge Nunc International, Rochester, NY, USA), or in 25-cm 2 culture flasks (Nalge Nunc International).The culture medium was changed twice a week, and the cells were used once they reached confluence (days 21 to 24).
The primary neuronal culture was prepared from the forebrain of fetal rats on embryonic day 16, as described previously [16].Assays were performed using cultures that were 7 to 10 days old.The nutrient medium remained untouched until the experiments were initiated.
Forty-eight hours before the assay, cilostazol was applied to cultured cells with or without KT5720, a potent inhibitor of PKA [17].Cilostazol exposure was typically started on day 5 -7 for the neurons and on day 19 -21 for the astroglia.To evaluate the acute effect of cilostazol on ROS generation, some cells were grown without cilostazol; instead, cilostazol was added to the H 2 DCFDA assay buffer during ROS measurement (see below).

Measurement of the Rate of L-[U-14 C]Lactate and [1-14 C]Pyruvate Oxidation to 14 CO 2
The rate of [ 14 C]lactate and [ 14 C]pyruvate oxidation to 14 CO 2 was measured using a modification of a previously described method [18].After the cells cultured in the 25-cm ]pyruvate (original concentration: 18.5 MBq/mL), which had been pre-warmed at 37˚C and equilibrated with 7% CO 2 to adjust the pH to 7.4, was added to the flasks.The culture flasks were then capped with rubber stoppers containing a center well and incubated at 37˚C for 60 minutes.The resulting 14 CO 2 was trapped by a cotton ball placed in the center well containing 100 μL of hyamine hydroxide 10-X.The reactions were terminated by the injection of 250 μL of 60% perchloric acid through the rubber stopper, and the flasks were kept at 4˚C overnight to trap the 14 CO 2 .The center wells were transferred to 20-mL glass scintillation counter vials, and 500 μL of ethanol and 10 mL of Insta-Fluor Plus were added.The 14 C contents of the vials were then evaluated using a liquid scintillation counter.
The cell carpets left in the incubation flasks after the removal of the reaction mixtures were then digested with 5 mL of 0.1 M NaOH, and their protein contents were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Meridian Road Rockford, IL, USA) based on the use of BCA for colorimetric detection [19], according to the manufacturer's instructtions.

Measurement of ROS Production
The production of ROS, mainly H 2 O 2 , in the cells was assessed using H 2 DCFDA [20] and semi-quantitative

Results
fluorometric measurements.Just prior to the assay, the nutrient medium was removed and the cells were washed twice with PBS without glucose.Then, DBSS containing 30 μM of H 2 DCFDA dissolved in dimethyl sulfoxide (DMSO) (final volume of DMSO: 0.1%) supplemented with 2 mM of glucose was added and the cells were incubated at 37˚C in humidified air with 7% CO 2 for 30 minutes.After loading the cells with the H 2 DCFDA, the cells were again washed twice with PBS without glucose, and DBSS containing 2 mM of D-glucose with cilostazol was added.The cells were further incubated for up to 60 minutes, and the fluorescent level indicating intracellular ROS production was measured at 0, 30, and 60 minutes using a fluorescent microplate reader (Cytofluor 4000 J, Applied Biosystems Japan, Tokyo, Japan) with excitation at 485 nm and emission at 530 nm.As the fluorescent signals increased linearly up to 60 min (data not shown), the results were expressed as the percent-increase in the fluorescent signal at 60 minutes over 0 minutes.When evaluating the acute effect of cilostazol on ROS production, ROS production was measured in the presence of cilostazol in some cells that had been cultured without the addition of cilostazol.

Cilostazol does Not Alter Astroglial or Neuronal Morphology
In the present study, we chose the concentration of cilostazol according to its clinical relevance.The usual dosage of cilostazol for post-ischemic stroke patients is between 100 -200 mg per day, and the maximal concentration of cilostazol in plasma is approximately 2 -5 μM (unpublished observations by manufacturer).Therefore, we applied cilostazol at concentrations of between 0.3 -30 μM in the culture medium.Dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), a membrane permeable cAMP analogue (0.5 mM), is well known to induce astroglial transformation, changing the cells from a cobblestone appearance to a fibrous shape and indicating cellular differentiation.After 48 hours of exposure to cilostazol, however, neither the astroglia nor the neurons exhibited any morphological changes, while dbcAMP (0.5 mM) induced astroglial transformation, with the cells assumeing a fibrous shape, and indicating cellular differentiation (data not shown).

Cilostazol Enhanced Both Lactate and Pyruvate Oxidation 2.6. Statistical Analyses
As shown in Figure 1, the rates of lactate oxidation were markedly enhanced both in neurons (right panel) and astroglia (left panel) after 48 hours of exposure to cilostazol.To confirm the enhancement of TCA cycle activity, we measured the activity of pyruvate dehydrogenase (PDH), which is a key enzyme that regulates entry to the TCA cycle by the rate of conversion of [ 14 C]pyruvate to 14 CO 2 .As depicted in Figure 2, PDH activation was observed in both neurons (right panel) and astroglia (left panel) at a concentration of 3 μM or over.
Statistical comparisons among the values obtained for each group (n = 3 or 4) were made using grouped t-tests or a one-way analysis of variance (ANOVA) followed by Dunnett's test for multiple group comparisons with a single control group, when applicable.A p-value of < 0.05 was considered statistically significant.For each experiment, at least two or three sets of assays were performed on different batches of cell preparations, and a set of representative data (mean ± SD) was presented in each figure or table.

Actions of Cilostazol Is Medicated by Protein Kinase a Pathway
To explore the downstream pathway that occurs in the presence of the cilostazol-mediated enhancement of glucose metabolism, we assessed the effect of dbcAMP.dbcAMP (0 -0.5 mM) induced astroglial morphological changes (data not shown) and also increased [ 14 C]lactate oxidation in astroglia (Figure 3), implying the involvement of cAMP.A highly specific PKA inhibitor, KT5720 (100 nM), abolished the cilostazol-induced (3 μM) increase in PDH activity in astroglia (Figure 4, left panel) and diminished that in neurons (Figure 4, right panel), indicating that cilostazol induces the oxidative metabolism of glucose through PKA-mediated PDH activation.

ROS Production in Neurons and Astroglia Was Not Altered by Cilostazol
Next, we evaluated whether the cilostazol-induced activation of the oxidative metabolism of glucose is associated with an augmentation of ROS production.Table 1 shows that ROS production in the neurons and astroglia was not affected, irrespective of the increased mitochondrial activity induced by incubation with cilostazol (0.3 -30 μM) for 48 hours.As cilostazol is reported to possess radical scavenging effects [10], we also evaluated the acute effect of cilostazol on ROS generation during an ROS assay.Table 1 shows that in neurons, lower concentrations of cilostazol (0.3 -3 μM) tended to decrease ROS production while the highest concentration (30 μM) enhanced ROS production (p < 0.01, ANOVA followed by Dunnett's r test for multiple comparisons).The astroglia did not exhibit any changes in ROS production when incubated with cilostazol.

Discussion
The present study revealed that the administration of cilostazol, a PDE3 inhibitor, at clinically relevant concentrations leads to the enhancement of the oxidative metabolism of glucose both in neurons and astroglia, implying that cilostazol may enhance ATP production in the brain and secure its function.This action of cilostazol seems to be mediated by intracellular cAMP elevation, PKA activation, and enhanced PDH activity.cAMP is reported to enhance Na + , K + -ATPase activity in peripheral nerves [21], and cilostazol prevents Na + , K + -ATPase activity from being reduced in diabetic model animals through the cAMP c mponent [22].Therefore, o Copyright © 2011 SciRes.PP  cilostazol might also enhance Na + , K + -ATPase activity in cultured neurons and astroglia by elevating intracellular cAMP levels, leading to an enhancement in the oxidative metabolism of glucose because Na + , K + -ATPase consumes approximately 50% of the total ATP generated in the brain during the resting state [1].Although we did not measure the Na + , K + -ATPase activity directly in the present study, our previous report showed that ouabain, an Na + , K + -ATPase inhibitor, reduced glucose utilization by about one half, as measured by evaluating [ 14 C]deoxyglucose phosphorylation [16].Therefore, cilostazol might enhance the oxidative metabolism of glucose by enhancing Na + , K + -ATPase activity.
The activation of Na + , K + -ATPase does not necessarily lead to an enhancement in the oxidation of glucose because ATP synthesis can only be provided by the enhancement of glycolysis without enhancing the oxidative metabolism of glucose.Astroglia, in particular, are more dependent on glycolysis for basal ATP production than neurons [18].However, we observed increases in lactate oxidation in both neurons and astroglia, clearly indicating that the oxidative metabolism per se is activated and that the primary enhancement of PDH activity is an un-derlying mechanism (Figure 4).In the brain, astrocytes play important roles in glucose metabolism.Glucose supplied from brain vessels might be taken up by astrocytes and metabolized to lactate through the glycolytic pathway, even in the presence of an adequate supply of oxygen (aerobic glycolysis).Lactate is thought to be transferred to neurons and oxidized through the TCA cycle; this pathway is known as the astrocyte-neuron lactate shuttle hypothesis (ANLSH) [23,24].If this hypothetical metabolic flow is correct, a simultaneous enhancement of glycolysis in astrocytes and oxidative metabolism in neurons would favor energy production in the brain as a whole.Importantly, astrocytic energy metabolism might vary between glycolytic and oxidative pathways in response to glucose environments.Under low glucose conditions, oxidative glucose metabolism in astroglia is activated and is almost comparable to that in neurons, as previously reported [18].As a matter of fact, the astrocytic oxidative metabolism of glucose, rather than glycolysis, is reported to be essential to memory function [25,26].Moreover, neuronal cell-specific glucose transporter-3 enables neurons to utilize glucose directly supplied from brain vessels without receiving lac-tate from astrocytes.Collectively, an enhancement in glucose oxidation in astrocytes might not necessarily worsen neuronal function.
The direct mechanism of the cAMP-dependent enhancement of PDH activity was not completely elucidated in the present study.PKA is a candidate enzyme that might regulate PDH activity because PKA phosphorylates various enzymes and regulates their action.PDH is regulated by PDH kinase, and the phosphorylation of PDH leads to inactivation [27][28][29].As shown in Figure 4, the PKA inhibitor enhanced the basal activity of PDH in both neurons and astroglia.However, the cilostazol-mediated action was not completely abolished in neurons, whereas it completely disappeared in astroglia.
Interestingly, we found that cilostazol did not enhance ROS production in either neurons or astroglia, irrespective of the enhanced oxidative metabolism of glucose.Of course, enhancing oxidative metabolism does not necessarily cause an immediate increase in ROS production.Brain tissue possesses an active anti-oxidant system to prevent oxidative stress, and failures in this system lead to various neurodegenerative diseases [30].Cilostazol reportedly has some action on the ROS scavenging effect [10].In fact, acute exposure to cilostazol somehow decreased ROS production in neurons and astroglia (Table 1).The exact mechanism of this effect is unclear.PKA and the productions of ROS are interrelated [31,32].Irrespective of the mechanism involved, cilostazol enhanced brain glucose metabolism without enhancing ROS production, which could have favorable effects on brain function.

Figure 3 .
Figure 3. Effects of 48 h of exposure to dibutyryl cAMP (0.05 -0.5 mM) on L-[U-14 C]lactate oxidation in astroglia.Values are the mean ± SD of quadruplicate wells.n.s., not significant, **p < 0.01 versus control (ANOVA followed by Dunnett's test for multiple comparisons).

Figure 4 .
Figure 4. Effect of 48 h of exposure to cilostazol (3 μM) on pyruvate dehydrogenase (PDH) activity ([1-14 C]pyruvate oxidation) in astroglia (left) and neurons (right) with or without exposure to a cAMPdependent protein kinase inhibitor (KT5720).Values are the mean ± SD of quadruplicate wells.n.s., not significant (ANOVA followed by Dunnett's test for multiple comparisons).