Báo cáo khoa học: cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells pdf

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.

The mechanisms involved in the compartmentaliza-
tion of long-chain acyl-CoA esters and free fatty acids
Keywords
acyl-CoA synthetase; acyl-CoA thioesterase;
arachidonic acid compartmentalization;
Leydig cells; steroidogenesis
Correspondence
E. J. Podesta
´
, Department of Biochemistry,
School of Medicine, University of Buenos
Aires, Paraguay 2155–5th, C1121ABG,
Buenos Aires, Argentina
Fax: +54 11 45083672 ext. 31
Tel: +54 11 45083672 ext. 36
E-mail:
(Received 27 July 2006, revised 23 August
2006, accepted 12 September 2006)
doi:10.1111/j.1742-4658.2006.05496.x
We have investigated the direct effect of arachidonic acid on cholesterol
transport in intact cells or isolated mitochondria from steroidogenic cells
and the effect of cyclic-AMP on the specific release of this fatty acid inside
the mitochondria. We show for the first time that cyclic-AMP can regulate
the release of arachidonic acid in a specialized compartment of MA-10
Leydig cells, e.g. the mitochondria, and that the fatty acid induces choles-
terol transport through a mechanism different from the classical pathway.
Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol trans-
port in isolated mitochondria from nonstimulated cells. The effect of arach-
idonoyl-CoA is inhibited by the reduction in the expression or in the
activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a

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-

tions [2]. Recently, using steroidogenic cells as an
experimental system, we described an alternative
releasing mechanism for AA as a mediator of hormone
action with the participation of an acyl-CoA synthe-
tase (ACS4) and a mitochondrial acyl-CoA thioest-
erase (Acot2) [7,8]. ACS4 has been described as an
AA-preferring acyl-CoA synthetase [9], while Acot2 is
a member of a new thioesterase family with long-chain
acyl-CoA thioesterase activity and it is associated with
the inner mitochondrial membrane [10–13].
In the steroidogenic cells, the step that determines
the rate of steroid synthesis (the rate-limiting step) is
the transport of cholesterol to the inner mitochondrial
membrane [14], a process in which ACS4 and Acot2
play a key role. In this mechanism, it has been sugges-
ted that ACS4 and Acot2 may constitute a system to
deliver AA into a specific intracellular compartment,
e.g. the mitochondria [8].
AA plays a crucial role in the steroidogenic cells,
mediating the induction of the steroidogenic acute reg-
ulatory (StAR) protein, one of the proteins involved in
cholesterol transport [15–19]. Although it is clear from
previous publications that AA plays its role in the pro-
cess through the conversion to its lipoxygenated
metabolites [15,17,20], a direct action of this fatty acid
on cholesterol transport into the mitochondria cannot
be ruled out.
In this context, a positive action of nonesterified
fatty acids on cholesterol metabolism has been des-
cribed in the mitochondrial membrane [21]. It was also

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

in this step are ACS4 [28] and StAR [29]. ACS4 works
in the release of AA, which, in turn, acts on StAR pro-
tein induction.
Because exogenous AA stimulates steroidogenesis in
cells, the first experiment was carried out to study the
direct effect of AA on cholesterol transport in the
absence of newly synthesized StAR protein. For this
purpose, MA-10 cells were incubated with exogenous
AA either with or without submaximal concentration
of 8-bromo, 5¢-cAMP (8Br-cAMP) in the presence or
absence of CHX. Progesterone production as measure-
ment of cholesterol transport was evaluated in the
culture media after 1 h of incubation, as described
in Experimental procedures (Fig. 1). Exogenous AA
alone stimulated progesterone production, reaching
50–60% of the maximal value obtained with 8Br-
cAMP (Fig. 1A). Submaximal doses of 8Br-cAMP in
combination with AA stimulated steroid production at
the same level as the stimulation produced by satur-
ating doses of 8Br-cAMP. 8Br-cAMP-stimulated pro-
gesterone production was completely abolished in the
presence of CHX. However, AA-induced steroid pro-
duction was only partially blocked by the protein syn-
thesis inhibitor. Again, CHX did not totally reduce the
synergistic effect of AA on steroid production, either
(Fig. 1A). Protein synthesis inhibition did not affect
progesterone production supported by the water-
soluble derivative of cholesterol, 22(R)-OH-cholesterol,
AA release in a specific compartment of the cells A. F. Castillo et al.
5012 FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS

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

incubated in the presence or in the absence of 8Br-
cAMP. After this incubation, free AA was measured in
the mitochondria, as described in the Experimental pro-
cedures. Figure 2 shows the uptake of AA into the mito-
chondria in basal and stimulated conditions. As can
-CHX
+ CHX
AA Control
8Br-cAMP
0.2 m
M + AA
8Br-cAMP
0.2 m
M
8Br-cAMP
0.5 m
M
A
b
b
a
a
a
a
b,c
b,c
0
2
4
6

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.

(a) P < 0.001 versus control cells without CHX treatment; (b)
P < 0.001 versus respective treated cells in absence of CHX; and
(c) P < 0.01 versus control cells treated with CHX.
B
17
16
15
4
3
2
1
0
Control
8Br-cAMP
A
AA
Nuclei
Mitochondria
Control
8Br-cAMP
***
a i
r
d n o h c o
t
i m
) s t i n u y r a r t i b r a ( A
i
e l
c

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

stained with iodine. Bars denote levels (in arbitrary units) of AA in
mitochondria and nuclei. Results are expressed as the mean ± SD
from three independent experiments. *** P < 0.001 versus control
mitochondria.
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 5013
clearly be seen, 8Br-cAMP increased the uptake of AA
3 times compared with nonstimulated conditions, with-
out changing the content in the nuclear fraction.
For the second approach, mitochondria from non-
stimulated MA-10 cells were isolated and incubated in
the presence of AA. Figure 3A shows that AA elicited
a stimulatory effect on cholesterol transport measured
as progesterone synthesis. As expected, this effect was
not affected by the addition of CHX previous to the
addition of AA. Nordihydroguaiaretic acid (NDGA),
an inhibitor of AA metabolism, had no effect on AA
action. Although NDGA is commonly used as lip-
oxygenase inhibitor, it is also able to inhibit Acot2
[11]. However, in this case, we only evaluated its action
on AA metabolism since the stimulation was per-
formed with AA. Other fatty acids, such as oleic and
arachidic acids produced a small, but not significant,
effect. The total steroidogenic capacity of the isolated
mitochondria was determined by the incubation with
the water-soluble derivative of cholesterol, 22(R)-OH-
cholesterol. Neither CHX nor NDGA affected this
total steroidogenic capacity.
As we have proposed [8] that the conversion of AA-
CoA to AA within the mitochondria may constitute a

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

of AA-CoA on steroid synthesis in mitochondria isola-
ted from non stimulated MA-10 cells where Acot2 was
a
b
0.15
0.05
0.00
Control
AA
Oleoyl-CoA
AA-CoA
0.25
0.20
0.15
0.10
0.05
0.00
B
)nietorpgm/gn(enoretsegorP
a
a,b
a
a
b
a
0.25
0.20
0.15
0.10
0.05

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-

cholesterol
Fig. 3. Effect of fatty acids, CoA derivatives of fatty acids, BPB,
NDGA and malonyl-CoA on progesterone production in isolated
mitochondria. Mitochondria were isolated from MA-10 cells and
preincubated for 5 min at 37 °C in the absence or in the presence
of 10 lgÆmL
)1
CHX or 100 lM NDGA (A) or 0.1 mM BPB, 100 lM
NDGA or 100 lM malonyl-CoA (C). Mitochondria were then incuba-
ted for 20 min with 200 l
M AA, oleic acid or arachidic acid or with
5 l
M 22(R)-OH-cholesterol (A); 200 lM AA, AA-CoA or oleoyl-CoA
(B); or 200 l
M AA-CoA (C). Mitochondria were pelleted by centri-
fugation and progesterone concentrations were measured in the
supernatants by RIA. Data are shown as progesterone production
(ngÆmg
)1
mitochondrial protein) in the incubation media. (A) Results
are expressed as the mean ± SD from six independent experi-
ments. *** P < 0.001 versus control. (B) Results are expressed as
the mean ± SD from three independent experiments. (a) P < 0.001
versus control mitochondria; (b) P < 0.01 versus AA treated mito-
chondria. (C) Results are expressed as the mean ± SD from three
independent experiments. (a) P < 0.001 versus control mitochon-
dria; and (b) P < 0.01 versus AA-CoA treated mitochondria.
AA release in a specific compartment of the cells A. F. Castillo et al.
5014 FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS
knocked down was significantly reduced compared

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

the postmitochondrial fraction of cells treated with this
compound (Fig. 5C). The same effect was observed in
cells where Acot2 expression was blunted (Fig. 6).
Discussion
In the present paper, we show for the first time that
cAMP can regulate the release of AA in a specialized
compartment of the cells, e.g. the mitochondria, and
that the fatty acid induces steroid synthesis through a
mechanism different from the classical pathway invol-
ving the stimulation of StAR protein expression. This
biological effect can be seen by the addition of exogen-
ous AA to intact MA-10 Leydig cells (300 lm)or
isolated mitochondria (200 lm). In determining the
Acot2
β
-tubulin
***
D
c
R
p
/
iVMC
cRp
/
-
i
VMC
sa
2

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

Acot2 antisense
***
***
AA-CoA
Control
cRp
/
i
V
MC
c
Rp
/
-
i
VMC
sa 2
t
o
cA
/2
toc
A
β
ni
l
u
b
u
t

-
)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

treated mitochondria from pRc ⁄ CMVi-transfected cells. In (D),
*** P < 0.001 versus mitochondria from respective-transfected
cells without AA-CoA treatment.
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 5015
effective concentration of AA, we also assess AA’s bio-
activity. Although in some cases concentrations
between 1 and 10 lm AA are sufficient to detect a bio-
logical effect [2], other effects require 100–300 lm [2].
However, at this high concentration, AA may not even
be in solution. Previous studies performed with con-
centrations of AA of the same magnitude as ours
(200 lm) observed either none [30] or a small but signi-
ficant [31,32] effect of exogenously added AA on basal
steroid synthesis in MA-10 and rat Leydig cells,
respectively. In the present paper, we show the stimu-
lation of steroidogenesis using 300 lm AA in the pres-
ence of albumin. These different results can be
explained by the fact that protein binding can increase
the overall concentration of fatty acids present in an
aqueous environment by effectively decreasing the
insoluble fraction [2]. Albumin, in particular, binds
specifically to fatty acids [33]. In our experiments, we
added albumin in the preparation of the AA solution,
which allowed us to detect a significant stimulatory
effect of exogenous AA on steroid production of non-
stimulated steroidogenic cells (50–60% compared with
cAMP). The stimulation of steroid synthesis by exog-
enously added AA was lower than the stimulation
reached by cAMP or 22(R)-OH-cholesterol. These

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

pRc ⁄ CMVi-Acot2 antisense cDNA plasmids as described in Fig. 5.
After 72 h, MA-10 transfected cells were labeled and stimulated as
described in Fig. 2. (A) Representative autoradiography showing AA
spots in mitochondrial fractions. (B) Autoradiography spots quantifi-
cation by densitometry. Bars denote levels (in arbitrary units) of AA
in mitochondria from MA-10 transfected cells treated with or with-
out 8Br-cAMP. Results are expressed as the mean ± SD from
three independent experiments. (a) P < 0.01 versus mitochondria
from control pRc ⁄ CMVi-transfected cells; and (b) P < 0.001 versus
mitochondria from 8Br-cAMP treated pRc ⁄ CMVi antisense trans-
fected cells.
b
a
0
4
8
12
16
20
A
Control
8Br-cAMP
8Br-cAMP
+ BPB
B
Control
8Br-cAMP
8Br-cAMP
+ BPB
AA

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;

(b) P < 0.05 versus mitochondria from 8Br-cAMP-treated cells. (C)
AA-CoA content in the postmitochondrial fraction. Data are shown
as
14
C-AA-CoA in cpm ⁄ mg protein in the postmitochondrial fraction.
Results are expressed as the mean ± SD from three independent
experiments. ***P < 0.001 versus control.
AA release in a specific compartment of the cells A. F. Castillo et al.
5016 FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS
effect in which steroid synthesis reaches maximal
activation (Fig. 1). This result agrees with that
obtained by other authors [35], who suggested that a
critical threshold of cAMP or cAMP-dependent pro-
tein kinase activation is required for the synergistic
effect of AA on cAMP-stimulated StAR protein
expression and steroidogenesis. The fact that the cells
treated with exogenous AA can be stimulated with
8Br-cAMP indicates that mitochondrial function
regarding the regulation of cholesterol metabolism
remains intact. Moreover, it is known that any distur-
bance or swelling of the mitochondria produces a total
loss of regulation, rendering mitochondria that pro-
duce full steroidogenesis. In our case, treatment with
AA also does not cause loss of the regulation of ster-
oid biosynthesis supported by 22(R)-OH-cholesterol
(data not shown). This explanation rules out the possi-
bility that AA or bovine serum albumin would have
effect on mitochondrial integrity. This consideration is
also valid when fatty acids or AA-CoA are used in iso-
lated mitochondria, as both treatments can still be

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

the first time that AA incorporation into a specific
subcellular compartment (the mitochondria) has been
shown as a consequence of the action of this second
messenger. Steroidogenic cells express Acot2 and also
a cytosolic isoform, Acot1, which is 92.5% homolog-
ous to the mitochondrial enzyme. We have ruled out
the possibility that mitochondria would uptake AA
produced by the action of Acot1 outside the mitochon-
dria, as part of our experiments were performed with
isolated mitochondria where Acot1 was not present.
Moreover, while the overexpression of Acot2 results in
an increase of hormone induced steroid synthesis [8],
overexpression of Acot1 does not produce this effect;
conversely, it produced a slight inhibition of the pro-
cess (data not shown). This last result supports the
notion that Acot2 is the thioesterase involved in the
release of AA inside the mitochondria.
Our model explaining how AA is released into the
mitochondria also supports the concept that the select-
ive 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 that might otherwise
share its biochemical effects. This is demonstrated by
the fact that when the mitochondria are stimulated
with other fatty acids, the response is lower than with
AA; however, there is a significant response of steroid-
ogenesis to a different Acyl-CoA (oleoyl-CoA, Fig. 4).
Thus, the specificity of the action is not due to the fatty
acid itself but to the acyl-CoA available to the mito-

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

the mitochondrial outer membrane. The homology
between the DBI and an acyl-CoA binding protein cer-
tainly enhances this possibility. The specific interaction
between DBI and its endogenous receptor, the PBR
located on the outer ⁄ inner mitochondrial membrane
contact sites [37,38], may direct the AA-CoA to this
organelle. Contact sites between the mitochondrial outer
and inner membrane could represent the microenviron-
ment for bringing the machinery together to transport
AA-CoA [37] into the mitochondria and facilitate AA
release, which in turn facilitates cholesterol transport.
How intramitochondrial AA could stimulate cholesterol
transfer from the outer to the inner mitochondrial mem-
brane can also be explained by the action of AA on the
membrane permeability in the contact sites. This sugges-
tion is also in line with experiments showing that AA
induces the specific membrane permeability in heart and
liver mitochondria by opening the mitochondrial per-
meability transition pore [39,40]. The pore is a multiple
protein complex located in the mitochondrial contact
sites [41] and, in mitochondria of steroidogenic cells, it
participates in cholesterol transport.
In the present paper, we demonstrate that 20–30% of
total steroid production can be elicited without the
necessity for StAR synthesis. This is in accordance with
the pathological situation where deletions or mutations
of StAR are detected in humans born with the steroid
deficiency disease, lipoid adrenal congenital hyperplasia
[17,20,42–44]. Disruptions of the StAR gene in mice
produce similar phenotypes [18,45]. The effect of these

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

transport, ACS4 and PBR play an important role
[23–25,47].
Experimental procedures
Materials
Fatty acid-free bovine serum albumin, AA, arachidic and
oleic acids, 4-bromophenacyl bromide (BPB), oleoyl-CoA,
malonyl-CoA, 8Br-cAMP, 22(R)-OH-cholesterol, cyclohexi-
mide (CHX) and Waymouth MB752 ⁄ 1 cell culture media
were purchased from Sigma Chemical Co. (St Louis, MO,
USA). Nordihydroguaiaretic acid (NDGA) and AA-CoA
were from Fluka (Buchs, Switzerland). Sera, antibiotics and
trypsin-EDTA were from Gibco-Life Technologies Inc.
(Gaithersburg, MD, USA). All other reagents were of the
highest grade available.
Cell culture
The MA-10 cell line is a clonal strain of mouse Leydig
tumor cells that produce progesterone rather than testoster-
one as the major steroid. The cells were generously provi-
ded by M. Ascoli, University of Iowa, College of Medicine
(Iowa City, IA, USA) and were handled as originally des-
cribed [48].
Cells were incubated in the presence or absence of
10 lgÆmL
)1
CHX for 30 min and then stimulated with 8Br-
cAMP (0.2 mm or 0.5 mm), 300 lm AA or 5 lm 22(R)-OH-
cholesterol in the culture medium containing 0.1% fatty
acid-free bovine serum albumin. Progesterone production
was measured by radioimmunoanalysis (RIA) [7], and data
are shown as progesterone production (ngÆmL

)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.

As indicated in each figure, inhibitors such as 10 lgÆmL
)1
CHX, 100 lm NDGA, 0.1 mm BPB or 100 lm malonyl-
CoA were added to the reaction mixture and preincubated
5 min prior to the addition of AA, AA-CoA or 22(R)-OH-
cholesterol.
After the incubation time, mitochondria were pelleted by
centrifugation 16 000 g for 15 min and progesterone con-
centrations were measured in the supernatants by RIA.
Data are shown as progesterone production per mg of
mitochondrial protein (ngÆmg
)1
protein).
[1-
14
C]Arachidonic acid incorporation
in MA-10 cells
MA-10 cells were labeled following a previously described
methodology [17], with minor modifications. [
14
C]-AA
(New England Nuclear, Boston, MA, USA; specific activity
53.0 mCiÆmmol
)1
) was added to the cultures in a concentra-
tion of 1 lCiÆmL
)1
per well (2 · 10
6
cells) in serum-free

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
[

14
C]-AA-CoA formation was evaluated by extraction from
the aqueous phase according to the literature [49].
Plasmid transfection
MA-10 cells were transiently transfected by electroporation
as previously described [8,50]. Transfection efficiency varied
from 40 to 50% and was estimated by counting fluorescent
cells transfected with pRc ⁄ CMVi plasmid [51] containing
the enhanced form of green fluorescent protein (EGFP) [8].
MA-10 cells were transfected either with pRc ⁄ CMVi plas-
mid containing the Acot2 antisense cDNA [8] or with the
empty vector as control. Approximately 72 h after transfec-
tion, cells were used as described in the respective figures.
SDS/PAGE and immunoblot assay
Mitochondrial proteins were separated by SDS ⁄ PAGE
(10% acrylamide gels) and electrophoretically transferred to
poly(vinylidene difluoride) membranes (Bio-Rad Laborator-
ies Inc., Hercules, CA, USA) as described previously [8].
Acot2 protein was detected using anti-Acot2 antibodies [11]
and immunoreactive bands were detected by chemilumines-
cence using enhanced chemiluminescence reagents (GE
Healthcare, Chalfont St Giles, UK).
Protein quantification and statistical analysis
Protein was quantitated by the method of Bradford [52]
using bovine serum albumin as a standard. Statistical ana-
lysis was performed by t-test or anova followed by the
Student–Newman–Kuels test.
Acknowledgements
This work was supported in part by National Research
Council (CONICET), University of Buenos Aires

(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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