Tài liệu Báo cáo Y học: Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells ppt

Uncoupling of protein-3 induces an uncontrolled uncoupling
of mitochondria after expression in muscle derived L6 cells
Danilo Guerini
1
, Elisabetta Prati
1
, Urvi Desai
2
, Hans Peter Nick
1
, Rolf Flammer
1
, Stephan Gru¨ ninger
1
,
Frederic Cumin
1
, Machael Kaleko
2
, Sheila Connelly
2
and Michele Chiesi
1
1
Metabolic and Cardiovascular Diseases, Novartis Pharmaceuticals Ltd, Basel, Switzerland;
2
Genetic Therapy Inc., Gaithersburg,
MD, USA
The uncoupling proteins (UCPs) are thought to uncouple
oxidative phosphorylation in the mitochondria and thus
generate heat. One of the UCP isoforms, UCP3, is abun-

dantly expressed in skeletal m uscle, the major thermogenic
tissue in humans. UCP3 has been overexpressed at high
levels in yeast systems, where it leads to the uncoupling of c ell
respiration, suggesting that UCP3 may indeed be capable of
dissipating the mitochondrial proton gradient. This effect,
however, was recently shown to be a consequence of the high
level of expression and incorrect folding o f the prote in and
not to its intrinsic uncoupling activity. In the present study,
we investigated the properties o f UCP3 overexpressed in a
relevant mammalian host system such a s the rat myoblast
L6 cell line . UCP3 was expressed in r elatively low levels
(< 1 lgÆmg
)1
membrane protein) with the help of an
adenovirus vector. Immunofluorescence microscopy of
transduced L6 cells showed that UCP3 was expressed in
more than 90% of the cells and that i ts staining pa ttern was
characteristic for mitochondrial localization. The oxygen
consumption of L6 cells under nonphosphorylating condi-
tions increased concomitantly with the levels of UCP3
expression. However, uncoupling was associated with an
inhibition of the maximal respir atory capacity of mito-
chondria and was not affected by purine nucleotides and free
fatty acids. Moreover, recombinant UCP3 was resistant to
Triton X-100 extraction under conditions that fully solubi-
lize m embrane bound proteins.
Thus, UCP3 can be uniformly overexpressed in the
mitochondria of a r elevant muscle-derived cell line resulting
in the expected increase of mitochondrial uncoupling.
However, our data suggest that the protein is present in a n

incompetent c onformation.
Keywords: uncoupling protein; mitochondria; respiration;
thermogenesis; aden ovirus.
In the last few years several novel proteins homologous to
thermogenin have been identified. Thermogenin is the
uncoupling protein (UCP) first identified in brown fat
mitochondria and is now referred to as UCP1. While the
role of UCP1 in thermoregulatory thermogenesis is undis-
puted, t here is still uncertainty concerning th e physiological
role of the other homologues. The ubiquitous UCP2 [1,2]
could play an important role in modulating insulin secretion
by b-cells [3,4], in mediating fever du ring infection [ 5] or, as
a general mechanism, in protecting cells from oxidative
stress by limiting the mitochondrial production of reactive
oxygen species [6]. Another protein belonging to the same
family, UCP3, has received much attention because of its
restricted expression in skeletal muscle [7,8], the major
thermogenic tissue in higher m ammals [9]. The uncoupling
properties of this protein, in fact, could explain the high level
of nonphosphorylating oxygen consumption of skeletal
muscle mitochondria. UCP3 is considered a promising
target for pharmacological intervention in the obese state, as
a controlled i ncrease in skeletal muscle thermogenesis could
safely correct for the energy imbalance. In addition to this
possible role in thermogenesis, it has been proposed that
UCP3, whose expression is strongly induced by free fatty
acids (FFAs), could facilitate or be beneficial when the
energy utilization in the muscles shifts from carbohydrates
to lipids [10].
To gather i nformation abou t t he potential r ole of U CP2

and UCP3, many laboratories h ave investigated their
properties after overexpression in various host cell systems
or in transgenic animals. The first indications that UCP2
and UCP3 could play a role in mitochondrial uncoupling
were o btained using yeast expression sys tems [1,2,11,12]. In
contrast to observations of UCP1 overexpression in yeast,
the uncoupling activity of UCP2 and UCP3 has not been
shown to be regulated by FFA or purine nucleotides [13,14].
Mammalian cell lines such as L6, C2C12 or human primary
muscle cells have also been used to analyse the properties of
UCP3 [15,16]. In these cells, UCP3 decreased the mito-
chondrial m embrane poten tial [15] but also caused changes
in substrate flow, such as a n increased l actate secretion [16].
UCP2 and UCP3 have also been overexpressed in an
insulinoma cell line and found to uncouple respiration in
association with increased lipid oxidation [17]. R econstitu-
tion experiments with recombinant UCPs have clearly
Correspondence to M. Chiesi, Metabolic and Cardiovascular Diseases,
Novartis Pharmaceuticals Ltd, 4000 Basel, Switzerland.
Fax: + 4 1 61 696 3783, Tel.: + 41 61 696 4 485,
E-mail:
Abbreviations: UCP, uncoupling protein; KO, knock-out; DMEM,
Dulbecco’s modified Eagle’s medium; m.o.i., multiplicity of infection;
DABCO, 2,4-diazabicyclo-(2,2,2)-octane; RU, Ru[dpp(SO
3
Na)
2
]
3
;

MTP, mitochondrial import stimulation factor; FFA, free fatty acid.
(Received 1 8 October 2001, revised 28 D ecember 2001, ac cepted 9
January 200 2)
Eur. J. Biochem. 269, 1373–1381 (2002) Ó FEBS 2002
shown that, similar to UCP1, UCP2 and UCP3 also
transport protons across lipid membranes [18,19], strongly
suggesting that their major physiological function is to
increase the mitochondrial proton leak. The role of UCP3 in
the control of energy homeostasis became apparent after the
generation of mice with a disrupted UCP3 gene [20,21]. The
knock-out (KO) animals did not show any evident obese
phenotype thus indicating that UCP3 does not play an
essential role in whole body basal energy expenditure in
rodents. However, oxidative phosphorylation in the muscles
of KO mice was found to be much more efficient and the
rate of AT P production markedly increased [ 22]. Therefore,
although the lack of UCP3 has strong effects on the
efficiency of oxidative phosphorylation, these latter effects
must be masked by the induction of compensatory mech-
anisms th at increase ATP consumption. Finally, transgenic
mice overexpressing UCP3 in skeletal muscle have been
generated and characterized [23]. The transgenic animals
had uncoupled muscle mitochondria and remained lean
despite their hyperphagia.
The properties of UCP2 and UCP3 overexpressed in
yeast mitochondria have been recently questioned [24,25].
Careful analysis has shown that only nonphysiological, high
concentrations of UCP2 and UCP3 i nduced the changes in
the mitochondrial p roton permeability previously observed.
These uncoupling effects were the result of overexpression

of the recombinant proteins leading to a compromised
mitochondrial integrity rather then to an intrinsic property
of the proteins. T his could e xplain the lack of r egulation by
free fatty acids and purine nucleotides reported by the
previous investigations [13,14]. Such artefacts might not be
restricted to yeast but could also occur in more relevant
expression systems such as m uscle-derived cell lines or even
in transgenic animals.
In the present study, we addressed this problem by
analysing the characteristics o f muscle-derived L 6 cells that
express U CP3 u nder the control o f the relatively weak rous
sarcoma virus prom oter. It was observed that, at expression
levels giving rise to an uncoupling phenotype, the mito-
chondrial respiratory activity became impaired, the mito-
chondrial membrane potential showed no sp ecific response
to FFA or nucleotides, a nd most of the U CP3 could n ot be
extracted by nonionic detergents. These results strongly
indicate that in mammalian cells, recombinant UCP3 is
expressed in a n on-native state. Therefore, extreme care is
necessary when interpreting the results of a forced overex-
pression of UCP3 in any host system.
EXPERIMENTAL PROCEDURES
Cloning of hUCP3 cDNA and construction
of the adenovirus shuttle plasmid
The h UCP3 cDNA was obtained from P. M uzzin (Univer-
sity of Geneva, Switzerland). T he h UCP3 cDNA in pBlue-
script II S K(+) plasmid (Stratagene, La Jolla, CA, USA)
was digested with SpeIandClaI, purified by gel electrophor-
esis and ligated into SpeI/ClaI-digested adenoviral shuttle
plasmid pAvS6alx [26] to generate pAvhUCP3lx.

pAvhUCP3lx contains a constitutive Rous Sarcoma V irus
(RSV) promoter, a 198-bp fragment containing the a deno-
virus serotype 5-tripartite leader s equence, the hUCP3
cDNA, a nd an SV40 early polyadenylation signal.
Construction and
in vitro
characterization
of recombinant adenovirus
The recombinant adenovirus encoding human UCP3
(Av3hUCP3) was c onstructed by a rapid vector generation
protocol using Cre recombinase-mediated recombination
[27] of two plasmids, one containing th e r ight hand portion
of the adenoviral vector genome and a lox site, and the other
plasmid, pAvhUCP3lx, containing the left portion of the
viral genome, the UCP3 expression cassette, and a lox site as
described previously [26]. Both plasmids and the C re-
encoding plasmid were cotransfected into AE1-2a cells
(A549 cells stably transfected with E1/E2a regions under the
control of d examethasone inducible promoters [ 28]). Vector
genome integrity was verified by viral DNA restriction
analysis. Vector concentrations were determined by spectro-
photometric analysis [29]. Titers are stated as particles per
mL. The control vector, Av3Null, was identical to the
Av3hUCP3 vector except that it lacked a transgene, but
retained the RSV promoter and SV40 poly(A)+ signal.
Cell culture and adenoviral infection
L6 rat skeletal muscle cells [30] were culture d in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal
bovine serum at 37 °C und er 5% CO
2

. C ells were seede d at
adensityof1.5· 10
4
cellsÆcm
)2
in order to reach 80%
confluency the next day. Then cells were infected with the
recombinant hUCP3 or control b- Gal viral st ocks at
different multiplicity of infection (m.o.i.) by keeping the
total final amount of adenovirus particles constant (i.e. 10
4
).
This was achieved by combining hUCP3 and the control
b-Gal adenovirus particles at t he moment of cells transduc-
tion. Infection o f L 6 cells with the adenovirus particles was
performed with the help of the transfection reagent Lipo-
fectamine P lus (Life Technologies) following the supplier’s
instruction. The e fficacy of infec tion f or varying viral loads
was determined by staining for b-Gal (data not shown).
Metabolic [
35
S]methionine labeling and membrane
preparation
L6 cells were infected with hUCP 3, b-Gal or empty
(Av3Null) recombinant aden oviruses for 48 h. The cu lture
medium was replaced with methionine-free MEM (Gibco-
BRL) and the cells were left at 37 °C for 20 min. Then the
cells were incubated with methionine-free M EM containing
120 lCiÆmL
)1

[
35
S]methionine (1000 CiÆmmol
)1
,Amer-
sham) for 2 h at 37 °C. After labeling, the cells were
washed twice with DMEM, resuspended in 200 lL10m
M
Tris/HCl, pH 8.0, 1 m
M
EDTA, 0.25 m
M
dithiothreitol and
disrupted by three cycles of freezing-thawing. The mem-
branes were sedimented at 20 800 g for 5 min (Eppendorf
centrifuge) and solubilized in 200 lL1%SDSin10m
M
Tris/HCl, pH 8.0, 1 m
M
EDTA. The samples were separ-
ated on a 10–15% SDS/polyacrylamide gel, stained with
Coomassie Brilliant Blue and dried, prior to exposure to
autoradiographic films (24–72 h).
Immunoblotting
Ten m icrogra ms o f protein we re s ep arate d o n 1 2. 5% S DS/
polyacrylamide gels a nd electroblotted to nitrocellulose
1374 D. Guerini et al. (Eur. J. Biochem. 269) Ó FEBS 2002
membranes (Bio-Rad Laboratories). Blots were block ed
with 3% BSA in NaCl/P
i

with 0.1% Tween-20 (NaCl/P
i
/
Tween) for 1 h and incubated overnight at 4 °Cwith
an affinity-purified rabbit anti-hUCP3 Ig (UCP3-2A,
Alpha Diagnostic International Inc.; 1 lgÆmL
)1
)orwithan
affinity chromatography-purified mouse anti-prohibitin Ig
(0.5 lgÆmL
)1
; NeoMarkers). B lots were washed with NaCl/
P
i
/Tween and exposed to horseradish peroxidase-conjugat-
ed second ary anti-(rabbit I gG) I g or a nti-(mouse I gG) Ig a t
a 1 : 10000 dilution in NaCl/P
i
/Tween for 1 h at room
temperature. Blots were washed again an d developed by
enhanced chemiluminescence using a standard kit (ECL,
Amersham Pharmacia Biotech.).
Immunofluorescence
Unless otherwise stated, all steps were performed at room
temperature in a humidified chamber. L6 cells
(1.5 · 10
4
cellsÆcm
)2
) were seede d on poly-

L
-lysine-coated
slides and they were i nfected 24 h l ater w ith the adenovirus
particles for hUCP3 and b-Gal, as described above. After
48 h, the cells were washed three times in NaCl/P
i
,thenfixed
in 4% (w/v) paraformaldehyde/NaCl/P
i
for 60 min. The
cells were w ashed four times in NaCl/P
i
and then i ncubated
for 60 min in NaCl/P
i
containing 0.1
M
glycine, pH 8.6.
After three washes with NaCl/P
i
, the cells were permeabi-
lized using 0.1% ( v/v) Triton X-100 in NaCl/P
i
for 3 min,
followed by four NaCl/P
i
washes. The slides were then
incubated in blocking buffer containing 5% (v/v) fetal
bovine serum, 0.1% (w/v) BSA, 5% (v/v) glycerol, and
0.04% NaN

3
in NaCl/P
i
for 60 min. The slides were overlaid
with anti-hUCP3 Ig (UCP3–2 A , Alpha Diagnostic Intl
Inc.) diluted 1 : 100 in blocking buffer and kept under gentle
rocking for 90 min at room temperatu re. After fi ve washes
(5 min each) in blocking buffer, the slides were incubated for
60 min with secondary antibodies [goat a nti-(rabbit IgG) Ig
coupled to A LEXA 594, Molecular P robes] dilut ed 1 : 100
in blocking buffer. The slides were washed twice in b locking
buffer, twice in NaCl/P
i
and finally mounted in a medium
containing 80% g lycerol, 2.5% 2,4-diazabicyclo-(2,2,2 )-
octane (DABCO) in NaCl/P
i
, pH 8 .0. The cells were
observed in an AXIOVERT 10 microscope (Carl Zeiss)
equipped with epifluorescence illumination using a 10x, 20x,
40x and 63x oil immersion plan-neofluor objective. Images
were collected with a cleavage coupled device (CCD) camera.
Extraction of membrane bound proteins
L6 cells were harves ted a nd membranes prepared as
described above. Proteins were extracted from the mem-
branes as described p reviously [31]. B riefly, the m embranes
were suspended in a buffer containing 3.6% Triton X-100,
1.2
M
ammonium acetate, 1 m

M
EDTA, 5 m
M
phenyl-
methanesulfonyl flu oride and 1 m
M
dithiothreitol at a
concentration of 3.6 mg detergent per mg protein. The
suspension was sonicated and incubated for 20 min at 0 °C.
The solubilized m aterial was separated by centrifugation a t
100 000 g for 20 m in.
Measurements of oxygen consumption
Measurements of oxygen consumption of L6 cells were
performed by using Ru[dpp(SO
3
Na)
2
]
3
(RU), a water
soluble oxygen sensor. The probe was synthesized as des-
cribed previously [32]. The principle of measurement was
based on fluorescence quenching of the sensor by oxygen
dissolved in the reaction medium. The quantum yield of the
fluorescence, was shown to be linear with oxygen concen-
tration as p redicted by the Stern–Volmer E quation. L6 cells
were cultured and transfected as described above. The
medium was r emoved an d c ells were washed with NaCl/P
i
.

Cells were trypsinized and s uspended i n a solution contain-
ing 5.5 m
M
glucose, 120 m
M
NaCl, 4 m
M
KCl, 1 m
M
KH
2
PO
4
,1m
M
MgSO
4
,1.3m
M
CaCl
2
,10m
M
Hepes,
pH 7.4 (medium A) supplemented with 10% fetal bovine
serum. Aliquots containing 1.5 · 10
6
cells wer e centrifuged,
resuspended in 100 lL medium A supplemented with
20 l

M
RU at 32 °C, an d added to a cuvette containing
1.9 mL o f t he same medium that had b een preincubated at
32 °C in a temperature adjustable cuvette holder of a
PerkinElmer LS50B fluorimeter. E xcitation wavelength was
470 nm and e mission was measured at 600 nm.
Measurement of membrane potential DY
The membrane potential was r ecorded using the fluorescent
probe 3,3¢-dihexyloxacarbocyanine iodide DiOC6 obtain ed
from Molecular Probes. L6 cells expressing either hUCP3 or
b-Gal or the combination of the two different types of
adenovirus particles were first subjected to digitonin treat-
ment in order to permeabilize the plasma membrane. This
was p erformed by incubating L6 cells at a concentration o f
1 · 10
6
cellsÆmL
)1
into Na Cl/P
i
containing 50 l
M
digitonin
on ice f or 5 min. Cells were washed once i n buffer A (1 m
M
EGTA, 2 m
M
MgCl
2
,5m

M
phosphate, 5 m
M
Hepes
pH 8.0, 20 m
M
sucrose, 20 m
M
mannitol, 120 m
M
KCl).
To ensure removal of endogenous substrates and tightly
bound nucleotides, permeabilized cells were subsequently
treated f or 30 min at room temperature by gently shaking
with Dowex 21K (Fluka) in 2 10 m
M
sucrose, 70 m
M
mannitol and 10 m
M
Hepes, pH 7.4. This procedure was
proven to be effective in removing tightly bound nucleotides
to UCP1 in isolated mitochondria [33]. Cells were then
incubated w ith 150 n
M
of DiOC6 in buffer A at pH 7.4 f or
15 min at room temperature. After a subsequent centrifuga-
tion step, the mitochondrial membrane potential of the
permeabilized cells resuspended in buffer A at pH 7.4
( 2 · 10

6
cellsÆmL
)1
) was measured at room temperature
using a n e xcitation a nd an emission wavelength pair of 485
and 530 nm.
RESULTS
The aim of this study was to evaluate t he properties of
hUCP3 e xpressed in a relevant system, such as the muscle
cell line L6, at a much lower level than in yeast. L6 cells have
lost the ability to express endogenous UCP3 (only trace
amounts o f UCP2 mRNA can be found) and display well
coupled mitochondrial respiration (see below). These char-
acteristics make L 6 myoblasts particularly suited for studies
aimed at analysing small changes in their mitochondrial
characteristics that m ight be induced by the overexpression
of recombinant h UCP3. I n p reliminary attempts to e xpress
hUCP3, stable and inducible expression systems have been
tried. None of these efforts could successfully produce levels
of hUCP3 e xpression sufficient to give a measurable change
Ó FEBS 2002 Uncoupling properties of UCP3 in L6 cells (Eur. J. Biochem. 269) 1375
in the r espiration pr operties of the cells. Transient transfec-
tion methods based on adenovirus particles proved to be
more successful. L6 myoblasts, however, were found to be
extremely resistant to adenoviral infection. Only after
application of viral particles in great number (> 1000
m.o.i.) in combination with a transfection agent such as
Lipofectamine Plus, an homogeneous infection o f the
majority of the cells could be achieved. This is illustrated
in Fig. 1 in w hich immunocytochemistry using anti-hUCP3

Ig was u sed to v isualize h UCP3 expression. In control cells
transduced with b-galactosidase recombinant adenovirus,
the antibody reaction produced only a faint background
staining. On the other hand, almost every cell transduced
with hUCP3 recombinant adenovirus showed a strong
signal after the treatment with the UCP3 antibody. Inter-
estingly, discrete subcellular s tructures with a punctuated or
slightly elongated appearance were visible, which strongly
resembled mitochondria (Fig. 1E). [
35
S]Methionine labell-
ing experiments were carried out to verify if UCP3 was
expressed without perturbing the normal p rotein expression
pattern of the L6 cells. Figure 2 shows that, in fact, the
protein expression patterns of cells infected with either
control (empty) viruses or hUCP3 expressing viruses w ere
identical except f or a single major protein band d isplaying a
molecular weight corresponding to that of the hUCP3.
Figure 3 shows the level of hUCP3 expression after
infecting L6 cells with increasing amounts of UCP3
recombinant adenovirus. The total number of virus particles
was kept constant (i.e. 10 000 mo.i.) by adding a corres-
ponding amount of control virus expressing the b-Gal
protein. Two days after infection, the respiratory character-
istics of L6 cells were analysed and then the level of
recombinant h UC P3 expressed was determined by Western
blotting (Fig. 3A, upper and medium panels). Quantifica-
tion was based on a standard curve obtained with recom-
binant hUCP3 expressed in inclusion bodies from E. coli
(Fig. 3B). As the purity of hUCP3 in the inclusion bodies

was  80% (as estimated by Coomassie Brilliant Blue
staining), and the crude mitochondrial preparation used in
the analysis was still contaminated by other membrane
fractions, the levels of hUCP3 per mg mitochondrial protein
given in F ig. 3B represent an underes timation o f t he actual
levels. In t he absence of hUCP3 expression, the addition of
the mitochondrial H
+
-ATPase inhibitor oligomycin
strongly reduced the oxygen c onsumption. As the oligomy-
cin-resistant portion of respiration reflects the level of
proton leak of mitochondria, the low l evel of respiration in
the presence of oligomycin of L6 cells showed that they were
very well coupled. The oligomycin resistant respiratory
activity of the cells could be strongly stimulated by the
addition of the protonophore carbonyl cyanide m-chloro-
phenyl hydrazone (CCCP) (Fig. 3A, upper panel). Stimu-
lation of oxygen consumption by CCCP was maximal at
concentrations between 0.5 and 1 l
M
while higher concen-
trations were inhibitory (not shown). In t he presence of an
uncoupler, such as C CCP, the respiratory activity o f
Fig. 1. Localization of hUCP3 in L6 cells by immunofluorescence. L6 cells were seeded at a density of 1.5 · 10
4
cellsÆcm
)2
.Infectionwascarriedout
24 h later by incubating the cells f or 6 h in the presence of the transfection r eagent Lipofectamine Plus combined w ith adenoviruses contain ing
hUCP3 or b-gal cDNA (hUCP3 and control cells, r espectively) at a multiplicity of infection of 10

4
. For details see t he Materials and methods
section. After 2 days cells were stained with specific hUCP3 antibodies (UCP3–2 A from Alpha Diagnostic Intl Inc.). (A,B) Control L6 cells infected
with b-Gal adenoviruses under phase contrast (A) and fluorescent light (B), respectively. Panel C and D show L6 cells infected with hUCP3
adenoviruses under phase contrast and fluorescent light, respectively. Panel E illustrates a single L6 cell infected with hUCP3 viral particles at higher
magnification.
1376 D. Guerini et al. (Eur. J. Biochem. 269) Ó FEBS 2002
mitochondria is pushed to i ts maximal c apability. While the
basal oxygen consumption of the cells expressing UCP3 was
not affected, the portion of respiration measured in the
presence of oligomycin increased concomitantly to the levels
of hUCP3 expressed (Fig. 3 upper p anel, black bars). This
suggested that hUCP3 increased the uncoupling of mito-
chondria. It was noted, however, that in cells expressing
UCP3 the maximal respiration levels obtained i n the
presence of CCCP were clearly reduced (Fig. 3 upper panel,
grey bars). The amount of the membrane protein prohibitin,
a marker of the mitochondria inner membrane, was f ound
to remain constant also in cells expressing the highest
amounts of hUCP3 thus indicating that the t otal number of
mitochondria/cell was not appreciably affected (Fig. 3A,
lower panel).
It has been previously reported that the bulk of hUCP3
expressed in yeast is aggregated. In fact, most of the
recombinant UCP3 remained insoluble after extraction with
high concentrations of nonionic detergents such as Triton
X-100 that normally fully solubilize UCP1 or other
membrane bound proteins [31]. We applied a similar
procedure to membranes isolated from L6 cells expressing
recombinant hUCP3. Prohibitin that is localized in the inner

membrane of mitochondria could be fully solubilized by the
extraction procedure (see Fig. 4). On the other hand, a
consistent portion of the recombinant h UCP3 was r esistant
to solubilization thus indicating that it was presumably in an
aggregated form.
The proton transport activity of recombinant hUCP3
refolded and reconstituted in proteoliposomes requires FFA
and is strongly inhibited by p urine nucleotides [18,19]. In an
attempt to analyse whether the activity of the recombinant
hUCP3 i n L 6 cells was also r egulated by these c ompounds,
we measured the membrane potential of mitochondria in situ
after selective p ermeabilization o f t he plasma m emb rane of
the cells with a m ild digitonin treatment. The mitochondrial
potential was analysed using the probe DiOC6, whose
fluorescence becomes quenched at h igh membrane
potentials. The left trace of Fig. 5A illustrates a typical
Fig. 2. [
35
S]Met pulse ex periment o f infected L6 cells. Infection of L6
cells with hUCP3 or Null adenovirus vectors was carried out as des-
cribed in the legend to Fig. 1. Infected cells were grown for 2 days and
then a [
35
S]Met pulse experiment was performed as described in the
Experimental procedure s section. Cells were harvested, lysed and
analysed on a 12.5% SDS/polyacrylamide gel. The dried gel was
exposed for 24 h to autoradiographic film. The amount of sample
loaded to ea ch lane s c orresponded t o 1 50 000 –200 0 00 c pm. L ane 1 ,
cells infected with Null adenovirus; lane 2, cells infected with UCP3
recombinant adenovirus. T he position of t he probable UCP3 band is

indicated o n the right side o f the pan el.
Fig. 3. Effect of hUCP3 expression on mitochondrial uncoupling. (A)
L6 cells were infected with hUCP3 adenoviruses at various multiplic ity
of infection (m.o.i.) as indicated. The total number of moi was kept
constant by adding correspondin g amounts of control viruses (b-G al).
After 2 days, the effect of hUCP3 ex pression on the mitochondrial
respiration w as investigated (upper panel). Cellular o xygen consump-
tion was m easured using the fluorescent oxygen sensor R uCP ( white
bars). O ligomycin (oligo), wa s used a t a co ncentration o f 10 lgÆmL
)1
to inhibit the portion o f the total cellular respiration co upled to
oxidative phosphorylation (black bars). T he uncoupler CCCP (1 l
M
)
was added after oligomycin to achieve maximal respiratory rates (grey
bars).The expression levels of hUCP3 (middle panel) and of the typical
inner-mitochondrial membrane mark er prohibitin (lo wer panel) were
assessed by Western blot analysis using specific antibodies. The graphs
represent mean values ± SEM (n ¼ 5). (B) Immunoreactivity of
recombinant hUCP3 from inclusion bodies u sed as calibration to
quantify hUCP3 expression levels.
Ó FEBS 2002 Uncoupling properties of UCP3 in L6 cells (Eur. J. Biochem. 269) 1377
control experiment s howing that, after d igitonin treatmen t,
endogenous substrates delivering NADH to the mito-
chondrial complex I were still sufficient to energise the
organelles. Addition of rotenone that blocks the u tilization
of NADH by complex I, was needed to fully depolarize
mitochondria. A subsequent addition of succinate that
delivers electrons directly to complex III thus bypassing
rotenone inhibition, re-energised mitochondria. Finally,

addition of the K
+
ionophore valinomycin fully depolarized
mitochondria. This control experiment showed that mito-
chondria remained functional after skinning of the c ells and
that sufficient endogenous small molecular weight compo-
nents were retained to support mitochondrial respiration.
As nucleotides could also remain trapped and inhibit the
intrinsic uncoupling activity of hUCP3, the digitonin treated
cells were extensively incubated with the resin Dowex-K21.
This extracting procedure was found to effectively remove
endogenous substrates so that, after about 30 min incuba-
tion, mitochondria were completely de-energised (see level
of right trace in Fig. 5A before the addition of rotenone and
succinate). Hence, this procedure that has been originally
developed t o s trip nu cleotides t ightly bound to UCP1 from
isolated brown fat mitochondria [33] could be applied also
to skinned cells. As Dowex treatment p resumably r emoved
most of the purine nucleotides from skinned L6 cells, the
effect of exogenously added GDP on the membrane
potential of mitochondria could be investigated. Figure 5B
shows t hat m mol c oncentrations of GDP did not affect the
membrane potential in both c ontrol cells and in cells
expressing hUCP3 (subsequent quantification gave values in
the order of 1 lghUCP3permgmembraneprotein).To
exclude the possibility that the recombinant hUCP3 was
inactive because t he cells have been s tripped also of
endogenous FFAs, lauric acid was added. Reconstitution
experiments h ave p reviously shown that l auric a cid i nduces
optimal activation of hUCP3 [18,19]. No effect on the

membrane potential of mitochondria by lauric acid in cells
expressing hUCP3 could be noticed. At concentrations
above 10–20 l
M
a drop in the membrane potential was
observed (see Fig. 5B). This effect, however, was identical in
both, control and UCP3 expressing cells, and was not
reverted by the subsequent addition of GDP.
DISCUSSION
Heterologous yeast expression systems have proven to be
very useful to study the properties of UCP1 [34]. Once
expressed in yeast mitochondria, the protein was found to
be fully functional and to be regulated by FFAs and
nucleotides, s imilarly to when expressed i n i ts native
location, the mitochondria of the brown adipose tissue.
Analogous strategies have been used to characterize the
function of two novel, recently discovered UCP1 homo-
logues such as UCP2 and UCP3. A general finding has been
Fig. 4. Extraction of prohibitin and hUVP3. L6 cells were transduced
with hUCP3 a denoviruses a t 1 0
4
multiplicities of infection, and wer e
harvested 2 days later. After the isolation of the membrane fraction ,
proteins were extracted in the presence of 3.5% Triton X-100 and
separated f rom the insoluble material b y centrifugation. The proteins
in the vario us fractions ( T, total b efore extraction; S, solubilized p ro-
tein; P, i nsoluble protein aggregates i n the pellet) we re then separated
by SDS/polyacrylamide gel electrophoresis. Prohibitin and hUCP3
were visualized using specific antibodies after Western blotting. F or
details see Experimental p rocedures section.

Fig. 5. Effect of hUCP3 on mitochondrial DY in skinned cells.
Recording of the m itochondrial membrane potential DY was
performed with t he flu orescent p robe 3,3 ¢-dihexyloxacarbocyanine
DiOC6. L6 cells expressing either hUCP3 or b-Gal (control) were
subjected to digitonin treatment in o rder to permeabilize the plasma
membrane. To ensure removal of endogenous su bstrates and tightly
bound nucleotides, permeabilized cells were treated for 30 min with
Dowex K21. Where indicated, additions were: rotenone 5 l
M
(rot),
succina te 5 m
M
(suc), GDP 0.5 m
M
,lauricacid50l
M
(LA), valino-
mycin 10 n
M
(val). (A) Control skinned cells before (left) and after
Dowex K21 treatment (right) (untreated and treated, respectively). (B)
Skinned c ells overexpressing hUCP3 or b-gal (control) were treated
with Dowex K 21 before measuring the membrane potential.
1378 D. Guerini et al. (Eur. J. Biochem. 269) Ó FEBS 2002
that these latter UCPs display much stronger uncoupling
effects than UCP1 on the yeast cells, while their activity does
not seem to be reg ulated by nucleotides [11–14,35]. The lack
of regulation of UCP2 and U CP3 was quite unexpected a s
both proteins share with UCP1 an highly conserved putative
nucleotide binding domain. Moreover, the proton transport

activity of UCP2 and UCP3, once refolded from inclusion
bodies and reconstituted in p roteoliposomes, w as shown to
require FFAs such as lauric acid [18,19] and to be highly
sensitive to purine nucleotides [19]. Recent s tudies from two
different laboratories have s hed some light on the possible
reasons underlyin g these controversial findings, by showing
that the expression of UCPs in yeast can lead to nonselective
damage of m itochondrial integrity. T his damage c aused a n
increase in the proton leak that is not regulated by, for
example, nucleotides [24,25]. Relatively low levels of
expression of UCP2 and U CP3 ( sublg per mg mitochond-
rial protein) are sufficient to cause unregulated uncoupling.
The ÔclassicalÕ U CP1 can b e e xpressed a t concentrations up
to 1 lgÆmg
–1
without causing any nonselective damages of
the mitochondria thereby retaining its physiological, regu-
lated function. However, when expressed at levels higher
than 10 lgÆmg
)1
, UCP1 was also found to promote a
nonregulated uncoupling of yeast mitochondria [36].
It remains unclear why UCP1 attains a native confor-
mation once expressed in yeast while UCP2 and UCP3 do
not. Our data strongly suggest that this phenomenon may
not be restricted to yeast but occurs also in other host cells.
In the present study, low to moderate levels of UCP3 were
expressed in the rat m uscle-derived L6 cell line. When the
expression of UCP3 was about 0.1–0.2 lgÆmg
)1

membrane
protein, i.e. a concentration similar t o that f ound in skeletal
muscle, no significant uncoupling could be detected. A clear
increase of the nonphosphorylating respiratory activity (i.e.
uncoupling) was apparent only at the highest levels of
expression that resulted in a reduction of the maximal
cellular respiratory capability (as measured in the presence
of a strong uncoupling agent such as CCCP). Since the
amount o f prohibitin per mg of membrane pr otein w as not
affected, one can presume that the expression of hUCP3 did
not influence the number of mitochondria per cell. It is likely
therefore that the lower maximal cellular respiration
reflected a general impairment of the mitochondrial func-
tion caused by the presence of a noncompetent hUCP3,
rather than a decrease in mitochondrial number. This
hypothesis is supported by the lack of a specific effect of
lauric acid and GDP, even at m
M
concentrations, on the
membrane potential of mitochondria in cells expressing
UCP3. This observation is very similar to what was
previously reported using p ermeabilized yeast cells expres-
sing UCP3 [25]. The membrane potential of mitochondria
in permeabilized yeast cells expressing UCP1, on the other
hand, was reported to be strongly inhibited b y FFAs and
the effects were fully reversed by GDP [25]. To obtain
further evidence t hat the UCP3 is not proper ly folded w hen
expressed in L6 cells, membrane proteins of the infected cells
were extracted with high concentrations of the nonionic
detergent Triton X-100. This procedure h as been proposed

to be a simple and stringent assay to evaluate the state of
proteins localized in the inner mitochondrial membrane
[31]. While hUCP3 was shown to be associated to the
mitochondria by immunocyto chemical staining, the protein
was resistant to solubilization by h igh nonionic detergents.
Another typical mitochondrial membrane protein such as
prohibitin was fully solubilized demonstrating the efficacy
of the extraction procedure.
What is the cause of the improper folding of UCP2 and
UCP3 into the mitochondria, when their expression is
forced in a host cell? A search for interacting proteins u sing
a yeast-two hybrid system revealed that the C-terminals of
UCP2 and UCP3 (but not that of UCP1) specifically
interact with members of the 14.3.3 protein family [37]. The
14.3.3 proteins are located in the cytosol where they regulate
various aspects o f cell p hysiology. The 14.3.3 proteins h ave
also been named mitochondrial import stimulation factors
(MSFs) because of their ability to chaperone the insertion in
the mitochondrial membrane of some of the anion trans-
porters. Specifically, MSFs have been shown to facilitate the
docking of the precursor proteins of the mitochondrial P
i
and the ADP/ATP carriers on the Tom70–Tom37 complex,
an import receptor localized on the outer mitochondrial
membrane. One could hypothesize th erefore that UCP1 is
inserted into the mitochondria without any special transport
mechanism w hile UCP2 and U CP3 m ight require a specific
import machinery. In the absence of sufficient amounts of
specific 14.3.3 proteins, UCP2 and UCP3 might not be able
to cross the intermembrane space and reach the inner

mitochondrial membrane in a properly folded state.
A recent in vivo experiments (based on NMR analysis)
comparing UCP3 K O with w ild-type m ice, has s hown that
the a mount of UCP3 exp ressed in skeletal muscle of wild-
type animals, i.e. about 50–100 ngÆmg
)1
mitochondrial
protein, strongly affects the efficiency of oxidative phos-
phorylation. It was therefore surprising that the expression
of similar amounts in L6 myoblasts did not cause any
relevant change in the proton leak. One could argue that,
even at the lowest expression levels, the protein cannot be
properly inserted into the mitochondria without a corres-
ponding coexpression of some specific ancillary protein(s).
Alternatively, it is possible that certain cofactors necessary
for the uncoupling are missing in the cell c ulture system. In
this respect, it i s relevant t o mention t hat UCP2 and UCP3,
in contrast to UCP1, fully rely on the presence of superoxide
anions to display their proton transport activity [38].
In conclusion, our data strongly suggest that, as observed
in yeast, overexpression of UCP3 in a muscle derived cell
line causes an increase in mitochondrial proton permeability
that is the result of improper folding and thus does not
represent a physiologically relevant function of the protein.
These findings imply that, r esults and phenotypes obtained
after overexpression of UCP3, and possibly also UCP2, in
any host system (i.e. not on ly cellular but also in tissue
systems and even in transgenic animals) should be inter-
preted with care. In support of this, it was recently shown
that the in vivo expression of UCP3 in transgenic mice

causes an artefactual uncoupling as well [39].
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