Pharmacology & Pharmacy, 2011, 2, 315-321
doi:10.4236/pp.2011.24040 Published Online October 2011 (http://www.SciRP.org/journal/pp)
315
Cilostazol Enhances Oxidative Glucose
Metabolism in Both Neurons and Astroglia
without Increasing Ros Production
Shinichi Takahashi*, Yoshikane Izawa, Norihiro Suzuki
Department of Neurology, Keio University School of Medicine, Tokyo, Japan.
R Received August 5th, 2011; revised September 13th, 2011; accepted September 25th, 2011.
ABSTRACT
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 pre-
sent 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-14C]lactat e
([14C]lactate) or [1-14C]pyruvate ([14C]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 in-
creases in [14C]lactate and [14C]pyruvate oxidation in both the neurons and astroglia. Dibutyryl cyclic AMP (0 - 0.5
mM) also increased [14C]lactate oxidation, indicating cAMP-mediated PDH activation. In contrast, free radical forma-
tion 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.
Keywords: Astrocyte, Cilostazol, Glucose, Lactate, Pyruvate Dehydrogenase
1. Introduction
Brain function is completely dependent on the oxidative
metabolism of glucose for ATP synthesis. The functional
activation of local brain lesions triggers ATP consump-
tion by Na+, K+-ATPase, which restores the trans-mem-
brane Na+ and K+ gradients in the local cells [1]. In con-
trast 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 ex-
periment has shown that two PDE inhibitors, enoximone
and milrinone, suppress glucose oxidation in isolated
cardiac myocytes [4] in accordance with Randle’s hy-
pothesis [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 [7-9]. It exerts a potent anti-
thrombotic 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 exam-
ined extensively. Cilostazol also plays a neuroprotective
role during acute [10] and subacute [11] phases of cere-
bral ischemia. This action is thought to depend on in-
creases 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) phos-
phorylation pathway, resulting in anti-apoptotic effects
[12,13]. Moreover, cilostazol is reported to scavenge
reactive oxygen species (ROS) [10]. In brain tissue, mi-
tochondria are believed to be a main source of ROS be-

Cilostazol Enhances Oxidative Glucose Metabolism in Both Neurons and Astroglia without Increasing Ros Production 316
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.
2. Materials and Methods
2.1. 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 Labora-
tory Animal Care and Use Committee of Keio Univer-
sity.
2.2. Chemicals
Chemicals and materials were obtained from the follow-
ing sources: L-[U-14C]lactate ([14C]lactate; specific activ-
ity, 4.84 GBq/mmol), [1-14C]pyruvate ([14C]pyruvate;
specific activity, 0.67 GBq/mmol), Insta-Fluor Plus and
hyamine hydroxide 10-X were all obtained from Perkin-
Elmer Life Sciences (Boston, MA, USA); Dulbecco’s
modified Eagle medium with or without glucose, penicil-
lin, and streptomycin were obtained from Life Technolo-
gies (Grand Island, NY, USA); defined fetal bovine se-
rum was obtained from HyClone Laboratories (Logan,
UT, USA); trypsin-EDTA was obtained from (Gibco-
BRL/Invitrogen, Grand Island, NY); 2’,7’-dichlorodihy-
drofluorescein diacetate (H2DCFDA) was obtained from
Molecular Probe Inc. (Eugene, OR, USA); and all other
chemicals were obtained from Sigma (St Louis, MO,
USA). Cilostazol was a kind gift from Otsuka Pharma-
ceutical Co. Ltd. (Tokushima, Japan). Please do not re-
vise any of the current designations.
2.3. Preparation of Cells
Primary astroglial cultures were prepared from the cere-
bral 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 high-
glucose medium (diluted 1:4), and placed in uncoated
24-well culture plates (Sumitomo Bakelite, Tokyo, Ja-
pan), 12-well culture plates (Nalge Nunc International,
Rochester, NY, USA), or in 25-cm2 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 re-
mained untouched until the experiments were initiated.
Forty-eight hours before the assay, cilostazol was ap-
plied 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 H2DCFDA
assay buffer during ROS measurement (see below).
2.4. Measurement of the Rate of
L-[U-14C]Lactate and [1-14C]Pyruvate
Oxidation to 14CO2
The rate of [14C]lactate and [14C]pyruvate oxidation to
14CO2 was measured using a modification of a previously
described method [18]. After the cells cultured in the
25-cm2 culture flasks were washed twice with phosphate-
buffered saline without CaCl2 and MgCl2 (PBS), 2.5 mL
of Dulbecco’s balanced salt solution (DBSS: 110 mM
NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 0.9
mM NaH2PO4, and 44 mM NaHCO3) supplemented with
2 mM of lactate and 2.5 μL (approximately, 0.85 μM) of
[14C]lactate (original concentration: 3.7 MBq/mL) or 2
mM of pyruvate and 2.5 μL (approximately, 20 μM) of
[14C]pyruvate (original concentration: 18.5 MBq/mL),
which had been pre-warmed at 37˚C and equilibrated
with 7% CO2 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 14CO2 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 14CO2. 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 14C 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 Rock-
ford, IL, USA) based on the use of BCA for colorimetric
detection [19], according to the manufacturer’s instruct-
tions.
2.5. Measurement of ROS Production
The production of ROS, mainly H2O2, in the cells was
assessed using H2DCFDA [20] and semi-quantitative

Cilostazol Enhances Oxidative Glucose Metabolism in Both Neurons and Astroglia without Increasing Ros Production 317
3. 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 H2DCFDA dissolved in dimethyl sulfoxide
(DMSO) (final volume of DMSO: 0.1%) supplemented
with 2 mM of glucose was added and the cells were in-
cubated at 37˚C in humidified air with 7% CO2 for 30
minutes. After loading the cells with the H2DCFDA, 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 produc-
tion, ROS production was measured in the presence of
cilostazol in some cells that had been cultured without
the addition of cilostazol.
3.1. 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 concen-
tration 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 as-
troglial transformation, changing the cells from a cobble-
stone appearance to a fibrous shape and indicating cellu-
lar differentiation. After 48 hours of exposure to cilosta-
zol, however, neither the astroglia nor the neurons exhib-
ited any morphological changes, while dbcAMP (0.5 mM)
induced astroglial transformation, with the cells assume-
ing a fibrous shape, and indicating cellular differentiation
(data not shown).
3.2. 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 cilosta-
zol. 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 [14C]pyruvate to
14CO2. As depicted in Figure 2, PDH activation was ob-
served 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 per-
formed on different batches of cell preparations, and a set
of representative data (mean ± SD) was presented in each
figure or table.
Figure 1. Effect of 48 h of exposure to cilostazol (0.3 - 3 μM) on L-[U-14C]lactate oxidation in astroglia (left) and neurons
(right). Values are the mean ± SD of quadruplicate wells. n.s., not significant, *p < 0.05, **p < 0.01 versus control (ANOVA
followed by Dunnett’s test for multiple comparisons).

Cilostazol Enhances Oxidative Glucose Metabolism in Both Neurons and Astroglia without Increasing Ros Production 318
Figure 2. Effect of 48 h of exposure to cilostazol (0.3 - 30 μM) on pyruvate dehydrogenase (PDH) activity, as measured by the
conversion of [1-14C]pyruvate to 14CO2 in astroglia (left) and neurons (right). Values are the mean ± SD of quadruplicate
wells. n.s., not significant, *p < 0.05, **p < 0.01 versus control (ANOVA followed by Dunnett’s test for multiple comparisons).
3.3. 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 glu-
cose metabolism, we assessed the effect of dbcAMP.
dbcAMP (0 - 0.5 mM) induced astroglial morphological
changes (data not shown) and also increased [14C]lactate
oxidation in astroglia (Figure 3), implying the involve-
ment of cAMP. A highly specific PKA inhibitor, KT5720
(100 nM), abolished the cilostazol-induced (3 μM) in-
crease in PDH activity in astroglia (Figure 4, left panel)
and diminished that in neurons (Figure 4, right panel),
indicating that cilostazol induces the oxidative metabo-
lism of glucose through PKA-mediated PDH activation.
3.4. ROS Production in Neurons and Astroglia
Was Not Altered by Cilostazol
Next, we evaluated whether the cilostazol-induced acti-
vation of the oxidative metabolism of glucose is associ-
ated with an augmentation of ROS production. Table 1
shows that ROS production in the neurons and astroglia
was not affected, irrespective of the increased mitochon-
drial 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 con-
centrations 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 astro-
glia did not exhibit any changes in ROS production when
incubated with cilostazol.
Figure 3. Effects of 48 h of exposure to dibutyryl cAMP
(0.05 - 0.5 mM) on L-[U-14C]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).
4. Discussion
The present study revealed that the administration of
cilostazol, a PDE3 inhibitor, at clinically relevant con-
centrations 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 cmponent [22]. Therefore, o

Cilostazol Enhances Oxidative Glucose Metabolism in Both Neurons and Astroglia without Increasing Ros Production319
Figure 4. Effect of 48 h of exposure to cilostazol (3 μM) on pyruvate dehydrogenase (PDH) activity
([1-14C]pyruvate oxidation) in astroglia (left) and neurons (right) with or without exposure to a cAMP-
dependent 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).
Table 1. Effects of chronic or acute exposure to cilostazol on ROS production in astroglia and neurons.
Cilostazol (M)
0 0.3 3 30
Astroglia 43.9 ± 2.9 41.4 ± 5.8 39.8 ± 4.7 39.0 ± 1.8
Chronic
exposure Neurons 55.8 ± 3.1 53.9 ± 2.0 49.2 ± 5.0 56.6 ± 6.8
Astroglia 33.0 ± 8.3 35.3 ± 2.7 34.5 ± 3.2 32.2 ± 2.7
Acute
exposure Neurons 57.2 ± 4.0 52.2 ± 3.7 49.8 ± 3.7 65.9 ± 9.9*
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 con-
sumes 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 pre-
sent study, our previous report showed that ouabain, an
Na+, K+-ATPase inhibitor, reduced glucose utilization by
about one half, as measured by evaluating [14C]deoxy-
glucose phosphorylation [16]. Therefore, cilostazol might
enhance the oxidative metabolism of glucose by enhanc-
ing Na+, K+-ATPase activity.
The activation of Na+, K+-ATPase does not necessarily
lead to an enhancement in the oxidation of glucose be-
cause ATP synthesis can only be provided by the en-
hancement 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 indicat-
ing 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 astro-
cytes 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 hy-
pothetical metabolic flow is correct, a simultaneous en-
hancement of glycolysis in astrocytes and oxidative me-
tabolism in neurons would favor energy production in the
brain as a whole. Importantly, astrocytic energy metabo-
lism might vary between glycolytic and oxidative path-
ways in response to glucose environments. Under low
glucose conditions, oxidative glucose metabolism in as-
troglia 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 glu-
cose transporter-3 enables neurons to utilize glucose di-
rectly supplied from brain vessels without receiving lac-

Cilostazol Enhances Oxidative Glucose Metabolism in Both Neurons and Astroglia without Increasing Ros Production 320
tate from astrocytes. Collectively, an enhancement in glu-
cose oxidation in astrocytes might not necessarily worsen
neuronal function.
The direct mechanism of the cAMP-dependent en-
hancement of PDH activity was not completely eluci-
dated in the present study. PKA is a candidate enzyme
that might regulate PDH activity because PKA phos-
phorylates various enzymes and regulates their action.
PDH is regulated by PDH kinase, and the phosphoryla-
tion of PDH leads to inactivation [27-29]. 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 astro-
glia.
Interestingly, we found that cilostazol did not enhance
ROS production in either neurons or astroglia, irrespec-
tive of the enhanced oxidative metabolism of glucose. Of
course, enhancing oxidative metabolism does not neces-
sarily 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 de-
creased 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]. Ir-
respective of the mechanism involved, cilostazol en-
hanced brain glucose metabolism without enhancing
ROS production, which could have favorable effects on
brain function.
5. Acknowledgements
The cilostazol used in this study was a kind gift from
Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan.
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