Uncoupling of protein-3 induces an uncontrolled uncouplingof mitochondria after expression in muscle derived L6 cells Danilo Guerini1, Elisabetta Prati1, Urvi Desai2, Hans Peter Nick1, R
Trang 1Uncoupling of protein-3 induces an uncontrolled uncoupling
of mitochondria after expression in muscle derived L6 cells
Danilo Guerini1, Elisabetta Prati1, Urvi Desai2, Hans Peter Nick1, Rolf Flammer1, Stephan Gru¨ninger1, Frederic Cumin1, Machael Kaleko2, Sheila Connelly2and Michele Chiesi1
1
Metabolic and Cardiovascular Diseases, Novartis Pharmaceuticals Ltd, Basel, Switzerland;2Genetic 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 muscle, the major thermogenic
tissue in humans UCP3 has been overexpressed at high
levels in yeast systems, where it leads to the uncoupling of cell
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 of the protein and
not to its intrinsic uncoupling activity In the present study,
we investigated the properties of UCP3 overexpressed in a
relevant mammalian host system such as the rat myoblast
L6 cell line UCP3 was expressed in relatively 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 its staining pattern 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 respiratory 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 membrane bound proteins
Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting
in the expected increase of mitochondrial uncoupling However, our data suggest that the protein is present in an incompetent conformation
Keywords: uncoupling protein; mitochondria; respiration; thermogenesis; adenovirus
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, there is still uncertainty concerning the 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 during 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 mammals [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 increase 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 information about the potential role of UCP2 and UCP3, many laboratories have 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 obtained using yeast expression systems [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 membrane potential [15] but also caused changes
in substrate flow, such as an increased lactate 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] Reconstitu-tion experiments with recombinant UCPs have clearly
Correspondence to M Chiesi, Metabolic and Cardiovascular Diseases,
Novartis Pharmaceuticals Ltd, 4000 Basel, Switzerland.
Fax: + 41 61 696 3783, Tel.: + 41 61 696 4485,
E-mail: michele.chiesi@phama.novartis.com
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 18 October 2001, revised 28 December 2001, accepted 9
January 2002)
Trang 2shown 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 ATP 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 that 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 induced the changes in
the mitochondrial proton 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 This could explain the lack of regulation 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 muscle-derived cell lines or even
in transgenic animals
In the present study, we addressed this problem by
analysing the characteristics of muscle-derived L6 cells that
express UCP3 under the control of the relatively weak rous
sarcoma virus promoter It was observed that, at expression
levels giving rise to an uncoupling phenotype, the
chondrial respiratory activity became impaired, the
mito-chondrial membrane potential showed no specific response
to FFA or nucleotides, and most of the UCP3 could not be
extracted by nonionic detergents These results strongly
indicate that in mammalian cells, recombinant UCP3 is
expressed in a non-native state Therefore, extreme care is
necessary when interpreting the results of a forced
overex-pression of UCP3 in any host system
E X P E R I M E N T A L P R O C E D U R E S
Cloning of hUCP3 cDNA and construction
of the adenovirus shuttle plasmid
The hUCP3 cDNA was obtained from P Muzzin
(Univer-sity of Geneva, Switzerland) The hUCP3 cDNA in
pBlue-script II SK(+) plasmid (Stratagene, La Jolla, CA, USA)
was digested with SpeI and ClaI, 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 Virus
(RSV) promoter, a 198-bp fragment containing the
adeno-virus serotype 5-tripartite leader sequence, the hUCP3
cDNA, and an SV40 early polyadenylation signal
Construction andin vitro characterization
of recombinant adenovirus The recombinant adenovirus encoding human UCP3 (Av3hUCP3) was constructed by a rapid vector generation protocol using Cre recombinase-mediated recombination [27] of two plasmids, one containing the right 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 Cre-encoding plasmid were cotransfected into AE1-2a cells (A549 cells stably transfected with E1/E2a regions under the control of dexamethasone 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 cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37°C under 5% CO2 Cells were seeded at
a density of 1.5· 104 cellsÆcm)2 in order to reach 80% confluency the next day Then cells were infected with the recombinant hUCP3 or control b-Gal viral stocks at different multiplicity of infection (m.o.i.) by keeping the total final amount of adenovirus particles constant (i.e 104) This was achieved by combining hUCP3 and the control b-Gal adenovirus particles at the moment of cells transduc-tion Infection of L6 cells with the adenovirus particles was performed with the help of the transfection reagent Lipo-fectamine Plus (Life Technologies) following the supplier’s instruction The efficacy of infection for varying viral loads was determined by staining for b-Gal (data not shown)
Metabolic [35S]methionine labeling and membrane preparation
L6 cells were infected with hUCP3, b-Gal or empty (Av3Null) recombinant adenoviruses for 48 h The culture 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 MEM containing
120 lCiÆmL)1 [35S]methionine (1000 CiÆmmol)1, Amer-sham) for 2 h at 37°C After labeling, the cells were washed twice with DMEM, resuspended in 200 lL 10 mM Tris/HCl, pH 8.0, 1 mMEDTA, 0.25 mMdithiothreitol 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 lL 1% SDS in 10 mM Tris/HCl, pH 8.0, 1 mMEDTA 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 micrograms of protein were separated on 12.5% SDS/ polyacrylamide gels and electroblotted to nitrocellulose
Trang 3membranes (Bio-Rad Laboratories) Blots were blocked
with 3% BSA in NaCl/Piwith 0.1% Tween-20 (NaCl/Pi/
Tween) for 1 h and incubated overnight at 4°C with
an affinity-purified rabbit anti-hUCP3 Ig (UCP3-2A,
Alpha Diagnostic International Inc.; 1 lgÆmL)1) or with an
affinity chromatography-purified mouse anti-prohibitin Ig
(0.5 lgÆmL)1; NeoMarkers) Blots were washed with NaCl/
Pi/Tween and exposed to horseradish
peroxidase-conjugat-ed secondary anti-(rabbit IgG) Ig or anti-(mouse IgG) Ig at
a 1 : 10000 dilution in NaCl/Pi/Tween for 1 h at room
temperature Blots were washed again and 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· 104cellsÆcm)2) were seeded on poly-L-lysine-coated
slides and they were infected 24 h later with the adenovirus
particles for hUCP3 and b-Gal, as described above After
48 h, the cells were washed three times in NaCl/Pi, then fixed
in 4% (w/v) paraformaldehyde/NaCl/Pi for 60 min The
cells were washed four times in NaCl/Piand then incubated
for 60 min in NaCl/Pi containing 0.1M glycine, pH 8.6
After three washes with NaCl/Pi, the cells were
permeabi-lized using 0.1% (v/v) Triton X-100 in NaCl/Pifor 3 min,
followed by four NaCl/Pi 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% NaN3in NaCl/Pifor 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 temperature After five washes
(5 min each) in blocking buffer, the slides were incubated for
60 min with secondary antibodies [goat anti-(rabbit IgG) Ig
coupled to ALEXA 594, Molecular Probes] diluted 1 : 100
in blocking buffer The slides were washed twice in blocking
buffer, twice in NaCl/Piand finally mounted in a medium
containing 80% glycerol, 2.5%
2,4-diazabicyclo-(2,2,2)-octane (DABCO) in NaCl/Pi, 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 harvested and membranes prepared as
described above Proteins were extracted from the
mem-branes as described previously [31] Briefly, the memmem-branes
were suspended in a buffer containing 3.6% Triton X-100,
1.2M ammonium acetate, 1 mM EDTA, 5 mM
phenyl-methanesulfonyl fluoride and 1 mM 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 material was separated by centrifugation at
100 000 g for 20 min
Measurements of oxygen consumption
Measurements of oxygen consumption of L6 cells were
performed by using Ru[dpp(SONa)] (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 predicted by the Stern–Volmer Equation L6 cells were cultured and transfected as described above The medium was removed and cells were washed with NaCl/Pi Cells were trypsinized and suspended in a solution contain-ing 5.5 mM glucose, 120 mM NaCl, 4 mM KCl, 1 mM
KH2PO4, 1 mM MgSO4, 1.3 mM CaCl2, 10 mM Hepes,
pH 7.4 (medium A) supplemented with 10% fetal bovine serum Aliquots containing 1.5· 106cells were centrifuged, resuspended in 100 lL medium A supplemented with
20 lM RU at 32°C, and added to a cuvette containing 1.9 mL of the same medium that had been preincubated at
32°C in a temperature adjustable cuvette holder of a PerkinElmer LS50B fluorimeter Excitation wavelength was
470 nm and emission was measured at 600 nm
Measurement of membrane potential DY The membrane potential was recorded using the fluorescent probe 3,3¢-dihexyloxacarbocyanine iodide DiOC6 obtained 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 performed by incubating L6 cells at a concentration of
1· 106cellsÆmL)1into NaCl/Picontaining 50 lMdigitonin
on ice for 5 min Cells were washed once in buffer A (1 mM EGTA, 2 mM MgCl2, 5 mM phosphate, 5 mM Hepes
pH 8.0, 20 mM sucrose, 20 mM mannitol, 120 mM KCl)
To ensure removal of endogenous substrates and tightly bound nucleotides, permeabilized cells were subsequently treated for 30 min at room temperature by gently shaking with Dowex 21K (Fluka) in 210 mM sucrose, 70 mM mannitol and 10 mM 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 with 150 nMof DiOC6 in buffer A at pH 7.4 for
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 · 106cellsÆmL)1) was measured at room temperature using an excitation and an emission wavelength pair of 485 and 530 nm
R E S U L T S
The aim of this study was to evaluate the properties of hUCP3 expressed 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 of UCP2 mRNA can be found) and display well coupled mitochondrial respiration (see below) These char-acteristics make L6 myoblasts particularly suited for studies aimed at analysing small changes in their mitochondrial characteristics that might be induced by the overexpression
of recombinant hUCP3 In preliminary attempts to express hUCP3, stable and inducible expression systems have been tried None of these efforts could successfully produce levels
of hUCP3 expression sufficient to give a measurable change
Trang 4in the respiration properties 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 of the
majority of the cells could be achieved This is illustrated
in Fig 1 in which immunocytochemistry using anti-hUCP3
Ig was used to visualize hUCP3 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 structures with a punctuated or
slightly elongated appearance were visible, which strongly
resembled mitochondria (Fig 1E) [35S]Methionine
labell-ing experiments were carried out to verify if UCP3 was
expressed without perturbing the normal protein 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 were
identical except for a single major protein band displaying 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 hUCP3 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 Fig 3B represent an underestimation of the actual levels In the absence of hUCP3 expression, the addition of the mitochondrial H+-ATPase inhibitor oligomycin strongly reduced the oxygen consumption As the oligomy-cin-resistant portion of respiration reflects the level of proton leak of mitochondria, the low level 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 lMwhile higher concen-trations were inhibitory (not shown) In the presence of an uncoupler, such as CCCP, the respiratory activity of
Fig 1 Localization of hUCP3 in L6 cells by immunofluorescence L6 cells were seeded at a density of 1.5 · 10 4
cellsÆcm)2 Infection was carried out
24 h later by incubating the cells for 6 h in the presence of the transfection reagent Lipofectamine Plus combined with adenoviruses containing hUCP3 or b-gal cDNA (hUCP3 and control cells, respectively) at a multiplicity of infection of 104 For details see the 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.
Trang 5mitochondria is pushed to its maximal capability 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 panel, 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 in 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 found
to remain constant also in cells expressing the highest
amounts of hUCP3 thus indicating that the total 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 hUCP3 was resistant
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 purine nucleotides [18,19] In an attempt to analyse whether the activity of the recombinant hUCP3 in L6 cells was also regulated by these compounds,
we measured the membrane potential of mitochondria in situ after selective permeabilization of the plasma membrane of the cells with a mild digitonin treatment The mitochondrial potential was analysed using the probe DiOC6, whose fluorescence becomes quenched at high membrane potentials The left trace of Fig 5A illustrates a typical
Fig 2 [ 35 S]Met pulse experiment of 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 [35S]Met pulse experiment was performed as described in the
Experimental procedures 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 each lanes corresponded to 150 000–200 000 cpm Lane 1,
cells infected with Null adenovirus; lane 2, cells infected with UCP3
recombinant adenovirus The position of the probable UCP3 band is
indicated on the right side of the panel.
Fig 3 Effect of hUCP3 expression on mitochondrial uncoupling (A) L6 cells were infected with hUCP3 adenoviruses at various multiplicity
of infection (m.o.i.) as indicated The total number of moi was kept constant by adding corresponding amounts of control viruses (b-Gal) After 2 days, the effect of hUCP3 expression on the mitochondrial respiration was investigated (upper panel) Cellular oxygen consump-tion was measured using the fluorescent oxygen sensor RuCP (white bars) Oligomycin (oligo), was used at a concentration of 10 lgÆmL)1
to inhibit the portion of the total cellular respiration coupled to oxidative phosphorylation (black bars) The 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 marker prohibitin (lower 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 used as calibration to quantify hUCP3 expression levels.
Trang 6control experiment showing that, after digitonin treatment,
endogenous substrates delivering NADH to the
mito-chondrial complex I were still sufficient to energise the
organelles Addition of rotenone that blocks the utilization
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 cells 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 to strip nucleotides tightly bound to UCP1 from
isolated brown fat mitochondria [33] could be applied also
to skinned cells As Dowex treatment presumably removed
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 that mmol concentrations of GDP did not affect the
membrane potential in both control cells and in cells
expressing hUCP3 (subsequent quantification gave values in
the order of 1 lg hUCP3 per mg membrane protein) To
exclude the possibility that the recombinant hUCP3 was
inactive because the cells have been stripped also of
endogenous FFAs, lauric acid was added Reconstitution
experiments have previously shown that lauric acid induces
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 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
D I S C U S S I O N
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, similarly to when expressed in its 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 adenoviruses at 10 4 multiplicities of infection, and were
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 from the insoluble material by centrifugation The proteins
in the various fractions (T, total before extraction; S, solubilized
pro-tein; P, insoluble protein aggregates in the pellet) were then separated
by SDS/polyacrylamide gel electrophoresis Prohibitin and hUCP3
were visualized using specific antibodies after Western blotting For
details see Experimental procedures section.
Fig 5 Effect of hUCP3 on mitochondrial DY in skinned cells Recording of the mitochondrial membrane potential DY was performed with the fluorescent probe 3,3¢-dihexyloxacarbocyanine DiOC6 L6 cells expressing either hUCP3 or b-Gal (control) were subjected to digitonin treatment in order to permeabilize the plasma membrane To ensure removal of endogenous substrates and tightly bound nucleotides, permeabilized cells were treated for 30 min with Dowex K21 Where indicated, additions were: rotenone 5 l M (rot), succinate 5 m M (suc), GDP 0.5 m M , lauric acid 50 l 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 cells overexpressing hUCP3 or b-gal (control) were treated with Dowex K21 before measuring the membrane potential.
Trang 7that these latter UCPs display much stronger uncoupling
effects than UCP1 on the yeast cells, while their activity does
not seem to be regulated by nucleotides [11–14,35] The lack
of regulation of UCP2 and UCP3 was quite unexpected as
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 proteoliposomes, was shown to
require FFAs such as lauric acid [18,19] and to be highly
sensitive to purine nucleotides [19] Recent studies from two
different laboratories have shed some light on the possible
reasons underlying these controversial findings, by showing
that the expression of UCPs in yeast can lead to nonselective
damage of mitochondrial integrity This damage caused an
increase in the proton leak that is not regulated by, for
example, nucleotides [24,25] Relatively low levels of
expression of UCP2 and UCP3 (sublg per mg
mitochond-rial protein) are sufficient to cause unregulated uncoupling
The ÔclassicalÕ UCP1 can be expressed at concentrations up
to 1 lgÆmg–1without 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 muscle-derived L6 cell line When the
expression of UCP3 was about 0.1–0.2 lgÆmg)1membrane
protein, i.e a concentration similar to that found 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 of prohibitin per mg of membrane protein was 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 mM concentrations, on the
membrane potential of mitochondria in cells expressing
UCP3 This observation is very similar to what was
previously reported using permeabilized 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 by FFAs and
the effects were fully reversed by GDP [25] To obtain
further evidence that the UCP3 is not properly folded when
expressed in L6 cells, membrane proteins of the infected cells
were extracted with high concentrations of the nonionic
detergent Triton X-100 This procedure has 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 immunocytochemical staining, the protein
was resistant to solubilization by high 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 using
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 of cell physiology The 14.3.3 proteins have 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 Pi and the ADP/ATP carriers on the Tom70–Tom37 complex,
an import receptor localized on the outer mitochondrial membrane One could hypothesize therefore that UCP1 is inserted into the mitochondria without any special transport mechanism while UCP2 and UCP3 might 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 KO with wild-type mice, has shown that the amount of UCP3 expressed 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 culture system In this respect, it is relevant to mention that 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, results and phenotypes obtained after overexpression of UCP3, and possibly also UCP2, in any host system (i.e not only 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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