cAMP increases mitochondrial cholesterol transport
through the induction of arachidonic acid release inside
this organelle in Leydig cells
Ana Fernanda Castillo, Fabiana Cornejo Maciel, Rocı
´
o Castilla, Alejandra Duarte, Paula Maloberti,
Cristina Paz and Ernesto J. Podesta
´
Department of Biochemistry, School of Medicine, University of Buenos Aires, Argentina
Arachidonic acid (AA) is a fatty acid with 20 carbons
and four cis double bonds that are the source of its
flexibility and its reactivity with molecular oxygen. The
oxidation can happen nonenzymatically or through the
action of three types of oxygenases: cyclooxygenase,
lipoxygenase and cytochrome P450. Most of the effects
of AA are attributable to its conversion by those
enzymes to prostaglandins, leukotrienes and other bio-
active products [1]. AA itself also has biological activ-
ity; however, the number of its described actions is
reduced compared to the effects described for the AA
metabolites. Moreover, it is not very well documented
whether nonmetabolized AA is released and elicits spe-
cial functions in a specific cellular compartment [2].
Transport of long-chain fatty acids in cells definitely
occurs when they are tightly linked to CoA by esterifi-
cation catalyzed by acyl-CoA synthetases [3]. In mam-
malian and yeast cells [4] it appears that the acyl-CoA
synthetases merely enhance uptake indirectly. Thus,
formation of the polar CoA-ester effectively traps the
fatty acid in the cell and functions as part of a facilita-
ted distribution in different cellular compartments.

substrate to release arachidonic acid. cAMP-induced arachidonic acid accu-
mulation into the mitochondria is also reduced when the mitochondrial
thioesterase activity or expression is blocked. This new feature in the regu-
lation of cholesterol transport by arachidonic acid and the release of
arachidonic acid in specialized compartment of the cells could offer novel
means for understanding the regulation of steroid synthesis but also would
be important in other situations such as neuropathological disorders or
oncology disorders, where cholesterol transport plays an important role.
Abbreviations
AA, arachidonic acid; AA-CoA, arachidonoyl-CoA; Acot2, mitochondrial acyl-CoA thioesterase; ACS4, acyl-CoA synthetase 4; BPB,
4-bromophenacyl bromide; 8Br-cAMP, 8-bromo-cAMP; CHX, cycloheximide; CPT1, carnitine-palmitoyl transferase 1; DBI, diazepam-binding
inhibitor; NDGA, nordihydroguaiaretic acid; P450scc, cholesterol side-chain cleavage cytochrome P-450 enzyme; PBR, peripheral
benzodiazepan receptor; StAR, steroidogenic acute regulatory protein.
FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS 5011
are important unresolved issues [5]. The simple struc-
ture of AA and the natural occurrence of so many
close chemical analogues are, not surprisingly, associ-
ated with a lack of specificity. The selective actions of
free AA may be explained simply by its specific release
under physiological conditions and by the absence of
such mechanisms for releasing other long-chain fatty
acids, compounds which might otherwise share its bio-
chemical effects [2]. Thus, the accessibility of AA to a
specific cellular compartment and the specificity of its
action are certainly linked.
The enzymes involved in the release of AA have
been well characterized, with the phospholipase A2
being the most important [6]. However, it remains
unclear as to how exactly AA is released in a specific
compartment of the cells under physiological condi-

demonstrated that cholesterol binding to the enzyme
that transforms it into pregnenolone (P450scc) in lipid
vesicles is greatly potentiated when the local membrane
is rendered more fluid by the addition of nonesterified
fatty acids [22].
All the evidence described above led us to propose
the hypothesis that AA might have a direct action on
cholesterol transport into the mitochondria via a speci-
fic release in this organelle. This knowledge would be
important for the understanding of cholesterol trans-
port in the classical steroidogenic as well as in neuro-
logical systems, since changes in cholesterol transport
in the central nervous system are part of the phenotype
seen in the neuropathology and neurological disorders
such as Alzheimer’s, Parkinson’s and Huntington’s dis-
eases, and brain injury and inflammation, as well as in
animal models of epilepsy [23]. This is also valid for
cholesterol transport and metabolism in tumors such
as glioma and mammary tumor cells [24,25].
For these reasons, the objective of the present work
was to study the release of AA into the mitochondria
and a possible direct role of fatty acids on cholesterol
transport in this organelle.
Results
It is known that the acute response of steroidogenesis
to hormonal stimulation has an absolute requirement:
de novo protein synthesis [26,27]. This conclusion is
based on the fact that hormone stimulated steroid syn-
thesis is totally inhibited by cycloheximide (CHX), a
protein synthesis inhibitor. The two proteins required

which travels freely across the membranes to reach the
inner mitochondrial cholesterol side-chain cleavage
cytochrome P-450 enzyme (P450scc) (Fig. 1B).
The widely known fact that cAMP cannot stimulate
steroidogenesis in the absence of protein synthesis is
due to the absence of two crucial proteins, ACS4 and
StAR. ACS4 is induced by hormones and it is neces-
sary for AA release [28], which participates in StAR
protein induction. Therefore, in the absence of ACS4,
no AA or StAR protein induction occurs, as previ-
ously described [28]. This is the reason why CHX com-
pletely abolished cAMP-stimulated steroidogenesis.
When exogenous AA is used in the presence of CHX,
the fatty acid bypasses the absence of ACS4 but not
the absence of StAR. Then, the partial inhibition pro-
duced by CHX on AA stimulated steroidogenesis was
unexpected. The stimulatory effect of AA on steroid
synthesis in the absence of protein synthesis suggests
that AA can per se enhance the cholesterol transport
and steroidogenesis in mitochondria of steroidogenic
cells without de novo protein synthesis. In order to test
this hypothesis, firstly, we tested whether AA exogen-
ously added to intact cells could reach the mitochon-
dria. Second, we studied the effect of exogenous AA
on cholesterol transport in isolated mitochondria from
nonstimulated MA-10 steroidogenic cells.
For the first approach, MA-10 cells were labeled with
[1-
14
C] AA during 5 h. After this period, the cells were

8
10
12
14
B
22(R)OH-
cholesterol
0
10
20
30
40
50
60
Progesterone (ng/ml)
Progesterone (ng/ml)
Fig. 1. Effect of cAMP, AA and CHX on progesterone production
by MA-10 cells. MA-10 cells were incubated in the presence or
absence of 10 lgÆmL
)1
CHX for 30 min and then stimulated for 1 h
with 8Br-cAMP (0.2 m
M or 0.5 mM) and ⁄ or 300 lM AA (A), or 5 lM
22(R)-OH-cholesterol (B) in serum-free culture medium containing
0.1% fatty acid-free bovine serum albumin. Progesterone concen-
trations were measured by RIA and data are shown as progester-
one production (ngÆmL
)1
) in the incubation medium. Results are
expressed as the mean ± SD from five independent experiments.

u n
a i r d n o
h
c
o t i m
i e l c u
n
Fig. 2. Effect of cAMP on mitochondrial and nuclear AA content.
MA-10 cells were labeled for 5 h at 37 °C with [1-
14
C] AA
(1 lCiÆmL
)1
per 2 · 10
6
cells) in serum-free media containing 0.5%
fatty acid-free bovine serum albumin. Then, cells were incubated in
either the presence or absence of 1 m
M 8Br-cAMP for 30 min.
After washing the cells, they were scraped and nuclear and mitoch-
ondrial fractions were obtained as described in the Experimental
procedures. The fractions were sonicated and lipids were extracted
with ethyl acetate. The organic phase was collected and dried
under nitrogen. The dried extracts were dissolved in chloro-
form:methanol (9 : 1, v ⁄ v) and analyzed by thin-layer chromato-
graphy on silica gel plates. (A) Representative autoradiography
showing AA spots in nuclear and mitochondrial fractions. (B) Auto-
radiography spots quantification by densitometry. The autoradio-
graphies were quantified by densitometry and the data were
normalized against the intensity of the signal of unlabeled AA

mechanism to deliver AA into specific compartment of
the cells, the next experiment was carried out to deter-
mine the effect of AA-CoA, the substrate of Acot2, on
steroid synthesis in isolated mitochondria. Figure 3B
shows that AA-CoA not only stimulated cholesterol
transport in isolated mitochondria but also had a
higher effect than free AA action. When another acyl-
CoA, such as oleoyl-CoA, was tested, it was also
proved capable of increasing progesterone production
in mitochondria to a similar extent.
To determine if the effect of AA-CoA on cholesterol
transport was due to its conversion to AA by the
action of Acot2, we studied the effect of the blockage
of Acot2 expression or activity on AA-CoA-stimulated
steroid synthesis. Our next experiment was conducted
as described in Fig. 3B in the presence and absence of
BPB or NDGA, both inhibitors of Acot2 activity [11].
Figure 3C shows that blockage of Acot2 activity pro-
duces a significant inhibition of progesterone synthesis
stimulated by AA-CoA.
To silence the expression of Acot2, we transiently
transfected MA-10 cells with pRc ⁄ CMVi plasmid con-
taining an antisense Acot2 cDNA (accession number
Y09333). The effect of antisense plasmid transfection
on Acot2 protein concentrations was studied by west-
ern blot, by means of a specific antibody against the
Acot2 and b-tubulin as control. As expected [8], anti-
sense-transfected cells showed a strong reduction in
Acot2 protein levels compared with cells transfected
with vector alone (Fig. 4A,B). The stimulatory effect

0.00
Malonyl-CoA
BPB NDGA
-AA-CoA
+ AA-CoA
Control
C
)nietorpg
m/gn
(enoretsegorP
b
bb
0.70
0.20
0.10
0.00
)nietorpgm/gn(enoretsegorP
0.85
Arachidonic
acid
Arachidic
acid
Oleic
acid
A
Control
+ NDGA
+ CHX
None
22(R)OH-

with mitochondria isolated from mock-transfected cells
(Fig. 4C). Acot2 knockdown did not produce any
effect on progesterone synthesis in mitochondria trea-
ted with 22(R)-OH-cholesterol (Fig. 4D). This provides
evidence that the reduction in Acot2 expression does
not affect mitochondrial integrity.
The results described above indicate the necessity of
Acot2 in AA-CoA-stimulated steroidogenesis, indica-
ting also that the effect of AA-CoA is due to its con-
version to AA into the mitochondria. If this is the
case, inhibition of AA-CoA uptake into the mitochon-
dria should inhibit steroid synthesis. Indeed, we inhib-
ited the carnitine-dependent acyl-CoA transport with
malonyl-CoA and the stimulatory effect of AA-CoA
on mitochondrial steroid synthesis was significantly
reduced (Fig. 3C).
The requirement of Acot2 for the action of AA-
CoA on steroid synthesis suggests the participation of
this enzyme in the mitochondrial cAMP-induced AA
accumulation. Then, we next tested the effect of Acot2
on mitochondrial [1-
14
C]-AA accumulation induced by
8Br-cAMP, using the same strategy described in
Fig. 3C and Fig. 4: inhibition of Acot2 activity and
expression, respectively. As shown in Fig. 5, BPB
inhibited cAMP induced accumulation of labeled AA
into the mitochondria (Fig. 5A,B). In accordance with
this effect produced by BPB on AA mitochondrial
content, there is an increase in AA-CoA retained in

to
cA
AB
22(R)OH-cholesterol
Control
pRc/CMVi pRc/CMVi-
pRc/CMVi pRc/CMVi-
Acot2 antisense
C
a
b,c
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
8
4
0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

-
)stinuyrartibra(
/
)nietorpgm/gn(enoretsegorPenoretsegorP
)nie
torpgm/gn(
Fig. 4. Effect of AA-CoA on progesterone production in isolated
mitochondria from Acot2 knockdown MA-10 cells. MA-10 cells
were transfected with pRc ⁄ CMVi or pRc ⁄ CMVi-Acot2 antisense
cDNA plasmids. After 72 h, mitochondria were isolated from MA-
10 transfected cells. (A) Representative western blot of mitochon-
dria from MA-10 transfected cells. The membrane was blotted
sequentially with anti-Acot2 and anti-b-tubulin sera. (B) Western
blot quantification by densitometry. Bars denote relative levels
of Acot2 expression in arbitrary units. *** P < 0.001 versus
pRc ⁄ CMVi transfected cells. (C) Mitochondria from MA-10 trans-
fected cells were incubated for 20 min at 37 °C in the absence or
in the presence of 200 l
M AA-CoA. (D) Mitochondria from MA-10
transfected cells were incubated for 20 min at 37 °C in the
absence or in the presence of 5 l
M 22(R)OH-cholesterol. In (C) and
(D), mitochondria were pelleted by centrifugation and progesterone
concentrations were measured in the supernatants by RIA. Data
are shown as progesterone production (ng ⁄ mg mitochondrial pro-
tein) in the incubation media. Results are expressed as the mean ±
SD from three independent experiments. In (C), (a) P < 0.001
versus control mitochondria from pRc ⁄ CMVi-transfected cells;
(b) P < 0.01 versus control mitochondria from pRc ⁄ CMVi-Acot2
antisense-transfected cells; and (c) P<0.01 versus AA-CoA

results are similar to those obtained by us in rat Ley-
dig cells and by other authors in the same or other
steroidogenic tissues [20,32,34].
When exogenous AA is added together with sub-
maximal doses of 8Br-cAMP, there is a synergistic
a
b
Acot2 antisense
B
AA
8Br-cAMP
Control
pRc/CMVi pRc/CMVi-
pRc/CMVi pRc/CMVi-
Acot2 antisense
A
a
b
0
1
2
3
4
5
6
7
control 8Br-cAMP control 8Br-cAMP
)stinuyrartibra(A
Fig. 6. Effect of Acot2 knockdown on AA accumulation into mito-
chondria. MA-10 cells were transfected with pRc ⁄ CMVi or

b
a
)stinuyrartibra(A
8Br-cAMP
+ BPB
0
25
Control 8Br-cAMP
[
14
C]AA-CoA
(cpm x10
–3
/mg protein)
C
50
***
Fig. 5. Effect of Acot2 activity inhibition on AA accumulation into
mitochondria and AA-CoA accumulation in the postmitochondrial
fraction. MA-10 cells were labeled as described in Fig. 2. When
indicated, cells were incubated with 0.1 m
M BPB for 30 min prior
to the stimulation with 8Br-cAMP. (A) Representative autoradiogra-
phy showing AA spots in mitochondrial fractions. (B) Autoradiogra-
phy spots quantification by densitometry. The autoradiographies
were quantified by densitometry and the data were normalized
against the intensity of the signal of unlabeled AA stained with iod-
ine. Bars denote levels (in arbitrary units) of AA in mitochondria.
Results are expressed as the mean ± SD from three independent
experiments. (a) P < 0.001 versus mitochondria from control cells;

increased by stimulation with 22(R)-OH-cholesterol
(data not shown).
As is already known [26,27], the cAMP-dependent
transport of cholesterol from the mitochondrial outer
to inner membrane can be blocked by a protein syn-
thesis inhibitor such as CHX. However, this protein
synthesis inhibitor is not totally able to abolish the sti-
mulation produced by exogenously added AA. These
results strongly suggest that AA can exert a role on
cholesterol transport without the induction of StAR
protein.
The demonstration that AA and ⁄ or AA-CoA stimu-
late cholesterol transport in isolated mitochondria sug-
gests that the accumulation of AA can occur by direct
uptake of AA itself inside the mitochondria or by the
previous esterification to AA-CoA by ACS4 and subse-
quent action of Acot2 to render free AA in the mito-
chondria. The fact that cAMP increases AA uptake
into the mitochondria and that this effect on AA accu-
mulation is reduced when Acot2 activity or expression
are blocked strongly indicates that the operating
mechanism is dependent on the concerted action
of ACS4 ⁄ Acot2. In this mechanism, cAMP acts to
increase AA-CoA formation in the cytosol. The CoA
derivative enters the mitochondria through the CPT1-
dependent pathway. The specificity of this mechanism
to release AA inside the mitochondria is shown by the
fact that the content of labeled AA in another organ-
elle such as the nucleus is neither increased by cAMP
nor reduced by the inhibition of Acot2 (Fig. 2). This is

chondrial Acot2. In our case, AA-CoA is formed pref-
erentially because of the specificity of ACS4 on AA [9].
The mitochondrial inner membrane is not permeable
to acyl-CoAs [3]; we wanted to know how AA-CoA
reaches the mitochondrial Acot2. The experiment using
malonyl-CoA (Fig. 5) indicates that AA-CoA follows
the usual pathway involving carnitine-palmitoyl transf-
erase 1 (CPT1) [3]. This enzyme plays a central role in
mitochondrial fatty acid oxidation. However, in our
case, it seems that CPT1 directs AA to another func-
tion. In this context, it has been proposed that a
potential route for long-chain acyl-CoAs to cross the
mitochondrial outer membrane could be the voltage-
dependent anion selective channel, also called mitoch-
ondrial porin and located in the contact sites [36]. It is
very interesting that a protein obligatory for choles-
terol transport in steroidogenic cells, the peripheral
benzodiazepine receptor (PBR), is also located in the
mitochondrial contact sites and includes the voltage-
dependent anion selective channel in its structure
together with the adenine nucleotide carrier [37]. PBR
is involved in cholesterol transport to the cytochrome
P450 side chain cleaving enzyme localized on the outer
surface of the mitochondrial inner membrane [37]. The
endogenous ligand of this receptor is an acyl-CoA
A. F. Castillo et al. AA release in a specific compartment of the cells
FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS 5017
binding protein known also as diazepam-binding inhib-
itor (DBI) [37,38]. It can be postulated that the role of
DBI is to facilitate the transport of fatty acids through

deletions establishes that StAR is necessary for 80–90%
of adrenal cholesterol metabolism [19,46]. In other
words, our results may explain the mechanism by
which in these situations there is a remaining 20% of
steroid synthesis, due to the direct effect of AA ⁄
AA-CoA produced within the mitochondria by the
action of ACS4 ⁄ Acot2 together with DBI ⁄ PBR.
Thus, it can be postulated that in the acute phase
(early response) of steroid synthesis, the release of AA
into the mitochondria is the first stimulator of choles-
terol transport. The sustained phase of the acute
response will then need the induction of StAR. We
cannot exclude that an extraordinarily small amount
of intramitochondrial StAR present in resting condi-
tions and not detectable by current techniques can
contribute to the effect of AA on cholesterol transport
in mitochondria.
The absence of hormone ⁄ cAMP-induced steroid
synthesis when protein synthesis is inhibited can be
explained now by the inhibition in the induction of
ACS4 [28] during the early response and the inhibi-
tion of ACS4 and StAR inductions during the sus-
tained phase. In both phases, the presence of
DBI ⁄ PBR may be necessary. This new feature in the
regulation of cholesterol transport by AA and the
release of AA in a specialized compartment of
the cells could offer novel means for understanding
the regulation of steroid synthesis, but would also be
important in other situations such as the neurosteroid
biosynthesis or oncology disorders, where cholesterol

)1
) in the
incubation medium.
Preparation of mitochondrial fraction
Mitochondria were obtained as previously described [17].
Briefly, all MA-10 cell cultures were washed with phos-
phate-buffered saline, scraped in 10 mm Tris ⁄ HCl (pH 7.4),
250 mm sucrose, 0.1 mm EDTA (TSE buffer), homogenized
with a Pellet pestle motor homogenizer (Kontes) and centri-
fuged at 800 g during 15 min. A second centrifugation at
16 000 g during 15 min rendered a mitochondrial pellet and
AA release in a specific compartment of the cells A. F. Castillo et al.
5018 FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS
a supernatant (postmitochondrial fraction). The mitochond-
rial pellet was resuspended in TSE buffer.
Progesterone production in isolated
mitochondria
Thirty microliters of mitochondrial fraction (200 lgof
protein) were added to 165 lL of medium consisting of
34 mm Tris ⁄ HCl (pH 7.4), 20 mm KCl, 4 mm MgCl
2
and
108 mm mannitol, containing 0.3% fatty acid-free bovine
serum albumin. When indicated, 200 lm AA, 200 lm AA-
CoA or 5 lm 22(R)-OH-cholesterol were added. The mix-
ture was completed by adding TSE buffer to complete a
final reaction volume of 500 lL (fatty acid-free bovine
serum albumin final concentration 0.1%). The incubations
were carried out at 37 °C for 20 min with gently shaking
and were stopped by cooling the tubes in an ice ⁄ water bath.

Waymouth MB752 ⁄ 1 containing 0.5% fatty acid-free
bovine serum albumin [17]. After 5 h of incubation at
37 °C in a humidified atmosphere containing 5% CO
2
, the
cells were incubated in the presence or absence of 1 mm
8Br-cAMP for 30 min. When indicated, cells were incuba-
ted with 0.1 mm BPB for 30 min prior to the stimulation
with 8Br-cAMP.
After these treatments, the cells were washed with serum-
free Waymouth medium containing 0.5% fatty acid-free
bovine serum albumin. Nuclear and mitochondrial pellets
were obtained as previously described [17] and resuspended
in 20 mm Hepes ⁄ KOH (pH 7.4), 250 mm sucrose, 1 mm
EDTA, 10 mm KCl and 1.5 mm MgCl
2
containing 500 ng
of unlabeled AA, and were then sonicated. Protein concen-
tration was measured and lipids were extracted from equal
amounts of nuclear or mitochondrial proteins (500 lgin
both cases) from each treatment. Lipid extraction was per-
formed twice with ethyl acetate (six volumes per one vol-
ume of nuclear or mitochondrial fraction). The organic
phase was then collected and dried under nitrogen at 25 °C
and analyzed by two successive thin-layer chromatographies
on silica gel. Radioactive spots were developed using a
Storm Phosphorimager (Amersham Biosciences, Sweden)
after 1 week of exposition. The postmitochondrial fraction
was treated as described for the mitochondria and the
[

(UBA) and National Agency of Scientific and Techno-
logical Promotion (ANPCyT). Thanks are due to the
technical assistance provided by F. Meuli.
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