 |
INTRODUCTION |
UCP21 belongs to a large
family of at least 35 anion carriers that are present in the inner
mitochondrial membrane (1). Most of these carriers transport key
metabolite substrates such as the malate, oxoglutarate, citrate, or
products from the oxidative phosphorylation such as ADP3
,
ATP4
, or Pi (for review see Ref. 2). Since
the discovery of ucp2 (3, 4) and ucp3 (5-7)
genes, a subfamily of mitochondrial carriers, related to the well known
UCP1 from brown adipose tissue, has emerged in mammals as well as in
plants (8). The deduced coding sequence for UCP2 predicts 59% identity
with UCP1, whereas the predicted UCP3 sequence is 72% identical to UCP2.
The common characteristic of these proteins is to uncouple the
respiratory chain from ATP synthesis by dissipating the proton electrochemical gradient when overexpressed in yeast mitochondria (for
reviews see Refs. 9 and 10). They may also transport anions, like the
other mitochondrial carriers, but these substrates are currently
unknown. Over the last few years, several physiological roles have been
proposed for UCP2, based on the expression of its mRNA upon various
physiological conditions (for review see Ref. 11). Genetic studies
suggested the novel UCPs might be linked to hyperinsulinemia (4) or to
the resting metabolic rate (12) and consequently to the control of body
weight. It was also proposed that UCP2 contributes to the inflammatory
response and regulates the production of reactive oxygen species from
mitochondria (13, 14). Recently, it has been shown that
ucp3(
/
) mice exhibited no consistent phenotypic
abnormality, despite a reduced proton leak in muscle mitochondria and a
higher level of intracellular ROS in muscle. Moreover, double knockout
ucp1-ucp3 phenotype was indistinguishable from the single
ucp1(
/
) phenotype (15, 16).
One of the main problems encountered in the physiological studies of
the new UCPs is the absence of reliable immunological tools to detect
the proteins in vivo. More than 100 publications have
described variation of UCP2 mRNA, whereas only seven analyses were
performed at the protein level (for reviews see Refs. 9 and 10). In the
present study, we have addressed this problem by generating antibodies
against the complete human UCP2 sequence, rigorously evaluated their
specificity and sensitivity, and compared them with commercial
antibodies. Mitochondria were prepared either from ucp2(+/+)
or ucp2(
/
) mice that were jointly created by the
Ricquier and Collins laboratories (17). UCP2 protein was immunodetected
without ambiguity in spleen, lung, stomach, and gonadal WAT (gWAT) but
not in other tissues. Although UCP2 protein was found in
vivo at a low level in spleen, lung, stomach, and WAT
mitochondria, its expression was strongly increased upon fasting and
LPS treatment in lung and stomach. Analysis of UCP2 mRNA and protein levels in transfected COS cells showed that the translation of
UCP2 transcript is strongly inhibited by an upstream ORF in the exon
two of the gene.
 |
MATERIALS AND METHODS |
Chemicals, Media, and Antibodies--
LPS, benzamidine,
aprotinin, pepstatin, leupeptin, bestatin, and phenylmethylsulfonyl
fluoride, CAPS, Tween 20, bicinchoninic acid kit, rabbit, sheep, and
goat horseradish peroxidase-conjugated antibodies were purchased from
Sigma. TPCK and mouse UCP2-IP antibody were obtained from Calbiochem.
Dulbecco's modified Eagle's medium, fetal calf serum, and
LipofectAMINE were purchased from Life Technologies, Inc. NHS-activated
Sepharose 4 fast flow and ECL detection kit were purchased from
Amersham Pharmacia Biotech. Human UCP2-NP antibody was obtained from
Research Diagnostic Inc., and mUCP22-A and hUCP32-A antibodies were
from Alpha Diagnostic International (San Antonio, TX), and
anti-cytochrome c antibody from Santa Cruz Biotechnology
(Santa Cruz, CA).
Animals and Treatments--
All mice were 7-10 weeks old.
ucp2(
/
) mice were generated on an mix inbred 129 and
C57BL/6J background (17). C57BL/6 mice were purchased from Elevage
Janvier (Orleans, France) and were submitted to 24 h of starvation
with free access to water or were injected intraperitoneally with 100 µg of LPS from Escherichia coli serotype 0111:B4 (4 mg of
LPS/kg of body weight). Control mice were injected with the same volume
of PBS. Mice were killed at different times by cervical dislocation.
Half of each organ was frozen in liquid nitrogen and kept for RNA
preparation, and the other half was immediately taken for the
preparation of fresh mitochondria.
Plasmids Construction--
The complete mouse UCP2 transcript
was obtained by PCR using the following primers, forward 5'
AAAATCAGTATGCGGCCGCCTTCTGCACTCCTGT 3' and reverse 5'
TTTCGCTCATTGCGGCCGCCGGGCTTTATGGGTG 3', and then subcloned in a
pcDNA3 vector (Invitrogen, Gronioen, Netherlands) after digestion
by NotI. The pUCP2-ORF1 was constructed by digestion with
the restriction endonuclease HindIII. Mutations of the
upstream initiator methionines at positions 123, 159, and 183 were
achieved with the Gene Editor kit (Promega, Charbonnières,
France) in combination with 2 sense primers 5'
GGACACAATAGTATCAACTTTAAGTGTTTC 3' and 5'
CCAGCCATTTTCTAGGGAAAATCGAGGGGATCGGGCCTTGGTAGCCACCGGC 3'. Mouse UCP2
cDNA was amplified by PCR using 5' GATCCATATGGTTGGTTTCAAGGCCAC 3'
and 5' ATGAAGCTTTCAGAAAGGTGCCTCCC 3' forward and reverse primers, respectively, and was cloned into NdeI and
HindIII restriction sites of pMW7 (18), a high copy number
expression plasmid closely related to the pET vector family (19).
Rat UCP1, human UCP2 and mouse UCP3 cDNA sequences were also
introduced into a pHis17 expression vector ((20) gift of M. Runswick, Dunn Nutrition Center, Cambridge, UK), a derivative of
pMW7 that contains six histidines in frame with BamHI,
HindIII, and EcoRI restriction sites of the
poly-linker. An internal NdeI site of the rat UCP1 cDNA
at nucleotide 581 was first disrupted by mutagenesis using the
following primer 5' GAGCTGGTGACGTATGACCTCATGAAGG 3' and subcloned into
NdeI and EcoRI sites of pHis17. Human UCP2 and
UCP3 cDNA were amplified by PCR using 5' GGATCCCATATGGTTGGGTTCAAGGC 3' and 5' AGCAAGCTTCCCCTTGTAGAAGGCTGTG 3' as forward primers and 5' CAGAAGCTTGAAGGGAGCCTCTCGGGA 3' and 5'
ATGGAACATATGGTTGGACTGAAGCC 3' as reverse primers. They were both cloned
into the NdeI and HindIII sites of pHis17.
Fragment of UCP2 cDNA encoding for UCP2-(95-206) peptide was
amplified by PCR using 5' GCATGCATATGCGCATTGGCCTCTACGAC 3' and 5'
ACTGGAATTCTTTCAGGAGAGTATCTTTG 3' forward and reverse primers,
respectively, and subsequently cloned into NdeI and
EcoRI sites of pHis17 expression vector. Cloning into the
HindIII of pHis17 expression vector added KLHHHHHH amino
acid sequence tag to the C terminus of the recombinant protein, and
cloning into the EcoRI site added EFHHHHHH sequence tag. All
constructs were sequenced on ABI 373A sequencer using the PRISM Bigdye
Terminator sequencing kit (PE-Applied Biosystems, Paris, France).
Preparation of RNA and Northern Blot Analysis--
Total RNAs
were extracted from frozen tissue as described previously (21) or from
transfected cells with a RNeasy kit (Qiagen, Courtaboeuf, France).
Northern analysis of 20 µg of total tissue RNA or 5 µg of total
cell culture RNA was carried out as described (22) using an
-32P-labeled and the complete mouse UCP2 cDNA as
probe (GenBankTM accession number U69135). Quantification
of UCP2 signal was determined with a Packard instant imager (Packard
Instrument Co., Meriden, CT), and the signal was normalized after
hybridization of the membrane with an 18 S rRNA probe.
Purification of UCP Proteins and Production of Anti-UCP2
Antibodies--
Fragments of mouse UCP2 and the full-length rat
UCP1, human UCP2, and mouse UCP3 were produced as inclusion bodies in
the E. coli C41 (DE3) bacterial strain and purified as
described previously (23). Proteins were refolded according to
Qiagen's protocol and purified in the presence of Fc12 detergent
(Anatrace, Maumee, OH) using nickel-nitrilotriacetic acid resin
(Qiagen, Courtaboeuf, France) and the Biologic Duo-flow high pressure
liquid chromatography system (Bio-Rad). Purified hUCP2 protein was
cross-linked to NHS-activated Sepharose 4 fast flow. Three hundred
micrograms of purified peptides or proteins were injected in 5 different rabbits, twice within 15 days and 1 month later. Animals were
bled 14 days after the last boost. Blood was left 1 h at room
temperature and then overnight at 4 °C and centrifuged at 3,000 × g. After inactivation of the complement at 55 °C for
20 min, serum was precipitated with ammonium sulfate according to Ref.
24 and purified by affinity chromatography on 2-ml hUCP2-NHS column as
described before (25).
Cell Culture and Transient Transfections--
The simian kidney
epithelial cell line, COS-7 cells, was routinely maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. Cells were transiently transfected using LipofectAMINE Plus
Reagent according to the manufacturer's instructions and harvested
24 h after transfection for UCP2 mRNA and protein analysis.
Isolation of Mitochondria--
All steps were carried out at
4 °C. Fresh tissues were minced in TES buffer (10 mM
Tris, pH 7.5, 1 mM EDTA, 250 mM sucrose) supplemented with the following protease inhibitors: 1 mM
benzamidine, 4 µg/ml aprotinin, 1 µg/ml pepstatin, 2 µg/ml
leupeptin, 5 µg/ml bestatin, 50 µg/ml TPCK, and 0.1 mM
phenylmethylsulfonyl fluoride. Minced tissue or COS cells were
carefully disrupted in a Thomas's potter at low speed rotation.
Unbroken cells and nuclei were removed by centrifugation of the
homogenate at 750 × g for 10 min. The supernatant was
centrifuged at 10,000 × g for 20 min, and the mitochondrial pellet was resuspended in 1 ml of TES buffer.
Mitochondria were submitted to another round of 10 min of
centrifugation at 750 and 10,000 × g, respectively.
Mitochondrial protein content was assayed by the bicinchoninic acid
method according to the manufacturer's protocol.
Western Blot Analysis--
Proteins were first separated on a
12.5% SDS-polyacrylamide and then transferred onto nitrocellulose
membrane by liquid electroblotting (Bio-Rad) for 75 min (125 mA per
gel) in a buffer containing 10 mM CAPS and 10% methanol,
pH 9. Nonspecific binding was achieved by preincubating the membrane
with PBS-T (phosphate-buffered saline containing 0.1% Tween 20)
supplemented with 5% dried milk for 1 h at room temperature. All
antibodies were diluted in PBS-T, 2% dried milk, and incubated
overnight at 4 °C. The concentration of the antibodies was carefully
optimized according to the signal to noise ratio. After extensive
washing of the membrane with PBS-T, appropriate peroxidase conjugate
antibody was incubated 1 h at room temperature in PBS-T, 2% dried
milk. Bound peroxidase-conjugated antibody was revealed with the
enhanced chemiluminescence reagents kit (ECL, Amersham Pharmacia
Biotech). Membrane was exposed for 1, 10, and 60 min on Biomax MR Kodak
film. Films were digitized by a Nikon Coolpix 950 camera, and signal
was quantified using the Image SXM software version 1.61 6P from
NCBI. For statistical analysis, samples were compared on the
same blot only.
 |
RESULTS |
Generation of Highly Sensitive UCP2 Antibodies--
Fragments of
the mouse UCP2 and the full-length rat UCP1, human UCP2, and mouse UCP3
were produced as inclusion bodies in the E. coli C41(DE3)
bacterial strain (23). Full-length human UCP2 and mUCP2 (amino acid
residues 95-206) proteins were purified on nickel columns in the
presence of Fc12 detergent and injected into rabbits. Three antisera
were obtained, two anti-hUCP2 sera and one anti-mUCP2 (95-206) serum.
Table I summarizes the data obtained with all UCP2 antibodies tested in this study. The rUCP1-375-5 serum, an antibody that we raised against the purified rat UCP1 (26),
displayed high sensitivity toward UCP1. This antibody could detect
only 80 ng of UCP2 inclusion bodies. The mUCP2-IP (Calbiochem),
hUCP32-A and mUCP22-A (Alpha Diagnostic) antibodies had a low titer and
poor reactivity toward UCP2. Among the anti-peptide antibodies, the
hUCP2-NP antibody (Research Diagnostic Int.) was the most sensitive
toward UCP2 and was 50 times more selective for UCP2 than either UCP1
or UCP3. However, mUCP2-2/3 and both antibodies we raised against the
full-length UCP2, hUCP2-605 and 606, displayed higher sensitivity
toward UCP2 than all the other antibodies (Table I).
In Vivo Distribution of UCP2--
Since the hUCP2-605 serum we
obtained was the most sensitive antibody and equipotent toward human or
mouse UCP2, it was selected to investigate UCP2 distribution in mouse
tissues. Western blot analysis revealed the presence of UCP2 protein in
spleen, stomach, intestine, lung, and white adipose tissue
mitochondria; this detection was specific since the 32-kDa band
detected by the antibody disappeared in mitochondria from
ucp2(
/
) mice (Fig.
1A). Surprisingly, we were
unable to detect UCP2 in muscle, heart, liver, and brain mitochondria.
Instead, other bands of similar apparent molecular weight appeared in
liver, BAT, and especially in brain mitochondria (Fig. 1B).
The strong band observed in BAT mitochondria appears to be UCP1 because
it disappeared in BAT mitochondria from ucp1(
/
) mice
(data not shown (27)). Incidentally, UCP2 did not appear in BAT
mitochondria from ucp1(
/
) mice although the
ucp2 gene is up-regulated in these mice (data not shown (27,
28)).

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Fig. 1.
Immunodetection of UCP2 in spleen, stomach,
intestine, lung, and gWAT. Mitochondria were prepared either from
ucp2(+/+) or ucp2( / ) mice tissues, and 30 µg of mitochondrial proteins were loaded onto an SDS-12.5% PAGE gel.
Western blot analysis was performed using the hUCP2-605 antibody at 0.1 µg/ml (10 min of exposure of the membrane). To confirm that the
equivalent amount of proteins were loaded, the same membrane was probed
with an anti-cytochrome c antibody. A, positive
control: 5 ng of mouse UCP2 inclusion bodies; immunodetection of UCP2
in spleen, stomach, intestine, lung, and gWAT. B,
immunoreactivity of the hUCP2-605 antibody in muscle, heart, liver,
brain, and BAT mitochondria.
|
|
Western blot analysis with hUCP2-606 and mUCP2-2/3 antibodies gave
similar results. However, both antibodies recognized more unspecific
proteins than hUCP2-605 antibody. All other antibodies tested in this
study (Table I) did not reveal any band in vivo and instead
some of them detected other proteins of similar molecular weight. The
hUCP2-NP antibody reacted strongly toward proteins in liver and kidney
mitochondria (Fig. 2A).
The rUCP1-375-5, our anti-UCP1 antibody, revealed a faint band in
spleen, liver, and, as expected, a strong band in BAT (Fig.
2B). The hUCP32-A antibody reacted with a 30-kDa protein in
kidney, liver, and BAT (Fig. 2C). None of these bands
disappeared in mitochondria isolated from ucp2(
/
) mice,
demonstrating that these antibodies are unable to detect bona
fide UCP2 in vivo.

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Fig. 2.
Tissue blot analysis with other anti-UCPs
antibodies. Mitochondria were isolated from tissues of
ucp2(+/+) mice or ucp2( / ) mice and analyzed
for their UCP2 content by Western blot. Gels were loaded with 30 µg
of mitochondrial proteins except for B where 3 µg of BAT
mitochondria were analyzed. Membranes were probed with different
antibodies. A, hUCP2-NP (Calbiochem, 1 µg/ml, 10 min of
exposure), the most sensitive anti-UCP2 peptide antibody; B,
rUCP1-375-5 (0.1 µg/ml, 60 min of exposure), our anti-UCP1 antibody
(26) that was reported to detect UCP2 by immunocytochemistry analysis
(40, 43); C, hUCP32-A (Alpha Diagnostic Int., 1 µg/ml, 10 min of exposure), an anti-UCP3 antibody that recognizes UCP2 as easily
as UCP3 (see Table I).
|
|
UCP2 Protein Is Expressed at Low Level in Vivo--
Quantitative
analysis showed that UCP2 was 4 and 10 times less abundant in lung and
stomach, respectively, than in spleen mitochondria (p < 0.001, Table II). To estimate the
amount of UCP2 protein expressed in vivo, we took advantage
of the cross-reactivity of hUCP2-605 antibody toward UCP1. As shown in
Fig. 3, 1 µg of BAT mitochondria
produced about the same signal in Western blot than 20 µg of spleen
mitochondria. The same experiment was repeated five times with
mitochondria from different mice. Quantification of the signals led us
to the conclusion that the hUCP2-605 antibody reacted 19-fold more
toward BAT mitochondria than spleen mitochondria (19 ± 2;
n = 5). Since the signal detected in BAT corresponded to UCP1 and given that the hUCP2-605 antibody was 8-fold more sensitive
toward UCP2 than UCP1 (Table I), we concluded that UCP2 is ~160-fold
less abundant in spleen mitochondria than UCP1 is in BAT
mitochondria.

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Fig. 3.
Quantitative comparison between UCP1 level in
BAT and the level of UCP2 in spleen mitochondria. Increasing
amount of spleen and BAT mitochondrial proteins were loaded on
SDS-12.5% PAGE. The membrane was probed with hUCP2-605 antibody at 0.1 µg/ml to detect UCP2 in spleen and UCP1 in BAT mitochondria (15 min
of exposure).
|
|
UCP2 Protein Is Increased in Lung and Stomach upon Fasting and LPS
Treatment--
It has been previously reported that fasting and LPS
treatment increased the level of UCP2 mRNA in skeletal muscle and
liver mitochondria, respectively (29-32). Therefore, C57BL/6 mice were either treated with LPS (injected intraperitoneally with 100 µg of
LPS or PBS buffer) or fasted for 24 h, and several organs were analyzed for their UCP2 protein content. Following 24 h of
fasting, UCP2 remained undetectable in muscle and constant in spleen
mitochondria, but a 6-fold increase of UCP2 protein was observed in
stomach and lung mitochondria (Fig. 4,
A and B). Two LPS-treated mice and two
PBS-treated mice were killed 6, 8, 10, 12, 14 (3 mice), 18, and 24 h after injection. Western blot analysis showed that UCP2 protein
appears 10 h after LPS injection only in lung mitochondria and
reaches its maximal level 2 h later. UCP2 protein returned to its
basal expression level 24 h after injection (Fig.
5, A and B). No
change in UCP2 protein was observed in the control mice treated with
PBS (Fig. 5B) and in stomach, liver, duodenum, kidney,
heart, muscle, and spleen of LPS-treated mice. Quantitative analysis of
LPS stimulation revealed that UCP2 protein increased 12-fold in lung
mitochondria 14 h after injection (p < 0.001, Table II). To assess whether the apparent increase of UCP2 protein in
lung mitochondria could simply reflect an increase of UCP2 protein
stability, 280 µg of cycloheximide, an inhibitor of protein translation, was also injected intraperitoneally in four mice 9.5 h after LPS injection. Two and one-half hours later all mice were
killed and analyzed for their UCP2 content in lung mitochondria. Cycloheximide treatment completely abolished UCP2 stimulation observed
12 h after LPS injection (98.2% inhibition p < 0.001, Table II). This experiment showed that LPS treatment stimulates the de novo synthesis of UCP2 protein in lung.

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Fig. 4.
Increase of UCP2 protein level in stomach and
lung upon fasting. Stomach (A) and lung (B)
mitochondria were isolated from three 24-h fasting mice and three
control mice. 30 µg of mitochondrial proteins were loaded on
SDS-12.5% PAGE and analyzed by Western blot. UCP2 protein was revealed
using hUCP2-605 antibody at 0.1 µg/ml. Northern blot analysis: RNA
(20 µg) were prepared from stomach (C) and lung
(D) of the same animals used for the preparation of
mitochondrial proteins. The full-length UCP2 cDNA was used as
probe. UCP2 signal was quantified after hybridization of the membrane
with 18 S rRNA probe.
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Fig. 5.
Induction of UCP2 protein in mouse lung
mitochondria after LPS injection. Mitochondria (30 µg) from two
LPS- or PBS-treated mice were prepared 6, 8, 10, 12, 14 (3 mice), 20, and 24 h after LPS injection. A, immunodetection of
UCP2 using hUCP2-605 antibody at 0.1 µg/ml (10 min of exposure).
B, graphic representation of UCP2 protein induction:
closed circle, LPS-treated mice; open circle,
PBS-treated mice. Level of UCP2 in lung mitochondria of noninjected
mouse was chosen as reference. C, Northern blot analysis:
total RNA (10 µg) were prepared from one lung of control mice or
LPS-treated mice. The full-length UCP2 cDNA was used as probe. UCP2
signal was quantified after hybridization of the membrane with 18 S
rRNA probe.
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|
UCP2 mRNA Levels Do Not Reflect UCP2 Protein Content in
Mitochondria--
To investigate whether the fluctuations in the
protein content correlated with gene expression, total RNAs were
prepared, and UCP2 mRNA level was estimated by Northern blot
analysis. Although UCP2 protein level was 4 and 10 times lower in lung
and stomach, respectively, than in spleen mitochondria, UCP2 mRNA
levels in those tissues were very similar (Table II). The same
discrepancy between UCP2 protein and mRNA levels was observed in
stimulated conditions. The levels of UCP2 mRNA in lung and stomach
marginally increased upon fasting (Fig. 4, C and
D, and Table II). UCP2 mRNA levels also remained
surprisingly unchanged over the LPS time course experiment (Fig.
5C). Fourteen hours after LPS injection, UCP2 mRNA
levels in spleen and lung were comparable, although UCP2 protein
content in lung had increased 12 times to reach three times the basal
level of UCP2 in spleen (Table II). Finally, despite the strong
variations of UCP2 protein that were observed in basal or stimulated
conditions, the level of UCP2 mRNA did not correlate with the
amount of UCP2 protein.
An Open Reading Frame in the Exon 2 of ucp2 Gene Inhibits the
Translation of the Protein--
The striking difference between the
abundance of UCP2 mRNA and the tiny amount of UCP2 protein detected
in mitochondria as well as the discrepancy between UCP2 mRNA and
protein levels prompted us to investigate whether expression was
regulated at the translational level. We have shown that
ucp2 gene contained a short open reading frame (ORF1) in
exon 2 which potentially encodes a putative peptide of 36 amino acids
(33) (Fig. 6A). Although the
translation of UCP2 protein was not affected by the ORF1 in
reticulocyte lysate (33), we examined whether its presence could
influence the expression of UCP2 in transiently transfected COS cells.
COS cells were transfected with the pcDNA3 expression plasmid,
encoding the complete mUCP2 cDNA sequence or the same construct
lacking the ORF1 sequence. The three methionine residues in ORF1 were
also mutated into serine residues (Fig. 6A). Due to the
polyadenylation tail of the pcDNA3 vector, recombinant UCP2
mRNA had a higher apparent molecular weight than mouse UCP2
mRNA (Fig. 6B). Although UCP2 mRNA level was 56 times higher in cells transfected with pUCP2 construct than in mouse
spleen, the amount of UCP2 protein was only 3.5 times higher in COS
cell mitochondria than in spleen mitochondria (Fig. 6B and
Table III). Thus, the coupling between
transcription and translation of ucp2 gene is more efficient
in mouse spleen than in transfected COS cells. However, transfection of
COS cells with pUCP2-ORF1 or pUCP2-ATG increased the production of UCP2 protein 50 and 17 times, respectively, in COS cell mitochondria, without major changes in UCP2 mRNA levels (Fig. 6, B and
C). In the absence of ORF1, UCP2 protein content in COS cell
mitochondria reached up to 176 times the level of UCP2 in spleen
mitochondria, although the UCP2 mRNA level had increased only 28 times (Table III). These results demonstrated that the ATGs present in
ORF1 strongly inhibits the translation of UCP2 mRNA and supports
the lack of correlation between UCP2 mRNA levels and protein
content observed in mouse tissues.

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Fig. 6.
An upstream ORF in exon two of the gene
inhibits the translation of UCP2. A, amino acid
sequence of ORF1 peptide is enlarged; the three methionines are marked
in bold and underlined. Roman numerals
indicate the exons in UCP2 cDNA inserted into pcDNA3 vector.
Punctual mutations of ATG codons in the pUCP2-ATG construct are
indicated in bold and underlined. B,
Northern blot and Western blot analysis of UCP2 expression after
transfection of COS cells. Upper panel, 20 µg of spleen
RNA and 5 µg of transfected COS cells RNA were analyzed by Northern
blot. Lower panel, Western blot analysis of UCP2 expression
was performed using the hUCP2-605 antibody (0.1 µg/ml, 1 min of
exposure) and using 30 µg of spleen mitochondria protein and 6 µg
of mitochondria protein from transfected COS cells. C,
graphic representation of UCP2 protein and mRNA levels. Cells
transfected with pUCP2 expression vector were chosen as references for
UCP2 protein and mRNA levels. Data are given as percentage of
pUCP2. Bar and error bar correspond to mean ± S.E.
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|
UCP3, BMCP1, and Other Mitochondrial Carriers Contain Upstream
ORF--
The 5'-untranslated region of all known mammalian
mitochondrial carriers was analyzed for the presence of upstream AUG or ORF. Among the 33 human and rodent cDNA sequences available, 9 of
them contained at least one upstream AUG or ORF. We found that ucp3 and bmcp1 also contained 1 and 3 ORFs,
respectively (Table IV).
Statistical parameters developed by Kochetov et al.
(34) were used to predict translational efficiency of these
mitochondrial carriers. Messenger RNA that are weakly translated
generally possess a long 5'-UTR (more than 50 nucleotides), a G + C
content in their 5'-UTR around 60%, and a context of complementary
nucleotides given by the A/U ratio between 0.75 and 1.25. In addition
to their upstream ORF, the five mitochondrial carriers that contained
upstream ORFs fulfilled at least two out of the three conditions
described above (Table IV). These results indicate that ucp3
and bmcp1 are probably subject to translational regulation
and, consequently, are poorly expressed in vivo.
 |
DISCUSSION |
Immunodetection of UCP2--
Several attempts were made to
identify in vivo UCP2 protein. Peptide antibodies (35-37),
antibodies against the full-length UCP2 (38), or anti-UCP1 antibodies
(14, 39, 40) were employed to detect the protein either by Western blot
or immunocytochemistry analysis (41-44). The identification of UCP1,
UCP2, or UCP3 was established by correlation with the tissue
distribution of the mRNA. It was also advocated that competition
with synthetic peptides and the apparent molecular weight confirmed the
identification of the bands as UCP1, UCP2, or UCP3 (38, 45). These
arguments are not sufficient to identify the UCPs in vivo
since most of the anti-UCP antibodies cross-react with other proteins.
This is especially true in liver mitochondria since four antibodies tested in this study detected nonspecific bands (Figs. 1 and 2). We
also tested the N-terminal UCP2 antibody (Santa Cruz Biotechnology) that has been used by Diehl's group (35, 37). One strong nonspecific band was detected in liver mitochondria around 32 kDa (data not show).
To reconcile biochemical data previously observed in liver and our
immunological data, one could speculate that there is another member of
the UCP family, specifically expressed in liver mitochondria, that
might have a similar function than UCP2. Our hUCP2-605 antibody also
shows a moderate specificity. Therefore, mitochondria from tissues of
ucp2(
/
) mice were essential to identify the protein
in vivo. By doing so, we demonstrated that UCP2 protein is
undoubtedly expressed in spleen, lung, stomach, and gWAT mitochondria.
Regarding the other tissues, UCP2 protein might be present at very low
levels, expressed only in some subset of cells or simply not expressed.
For instance, although UCP2 mRNA has a neuronal localization by
in situ hybridization, its distribution is not uniform in
the brain, and the mRNA is especially abundant in the
suprachiasmatic nucleus and other nuclei (46).
Translational Regulation of UCP2 Expression--
Several lines of
evidence support the possibility that UCP2 mRNA does not
correlate with the variation of the protein. First, UCP2 protein is 10 times less expressed in stomach mitochondria than in spleen even if the
relative amount of UCP2 mRNA in both organs is similar (Table II).
Second, the increase of UCP2 protein in lung and stomach upon fasting
or LPS treatment is not accompanied by an increase of UCP2 mRNA.
Third, inactivation of ORF1 in ucp2 gene increased 50 times
the expression of UCP2 protein in transfected COS cells without any
major changes at the mRNA level. Cotransfection of COS cells with a
vector encoding ORF1 peptide and pUCP2-ATG did not repress the
expression of the UCP2 protein, and disruption of the first or the
second ATG of the upstream ORF also stimulated the expression of UCP2
(data not shown). Thus, the ucp2 gene is down-regulated in
cis at translational level by an upstream ORF. These results
do not exclude transcriptional regulation of the ucp2 gene.
UCP2 mRNA is highly expressed in spleen, lung, stomach, and WAT but
is weakly expressed in the other tissues. Since UCP2 protein was found
only in tissues where the its mRNA was highly abundant, it is
possible that the tissue specificity of ucp2 gene expression
is partially controlled at the transcriptional level, whereas the level
of UCP2 protein is modulated by translational regulation. The molecular
mechanism by which UCP2 protein synthesis is suddenly stimulated
in vivo remains unclear. Reverse transcriptase PCR analysis
(between exon 1 and 4) during the LPS time course experiment did not
reveal an alternative splicing of UCP2 exon 2 explaining the increase
of UCP2 protein in lung (data not shown). Therefore, it is likely that
most of the ribosomes initiate the translation at the first ORF1 AUGs,
and only few of them initiate the translation at the downstream UCP2 AUG.
It is intriguing that all newly discovered UCPs also contain upstream
ORF. Since a discrepancy between mRNA and protein expression has
been also suggested for UCP3 by Sivitz et al. (45), one should be very cautious about mRNA variations of UCP2, UCP3, or BMCP1. For instance, Cadenas et al. (47) and Matthias
et al. (28) conducted extensive bioenergetic studies on
starved rat muscle mitochondria and BAT mitochondria from UCP1-ablated
mice, respectively. They showed that although ucp2 gene
transcription was strongly up-regulated, mitochondrial proton
conductance remained unchanged. In our hands UCP2 protein remained
undetectable in the BAT of Ucp1(
/
) mice. Given
the translational regulation of ucp2 gene, it is possible
that UCP2 mRNA levels do not always reflect the expression of the
protein itself.
Physiological Aspects--
It is admitted that UCP1 is a slow
proton transporter highly expressed in brown adipose tissue
mitochondria. In cold-adapted rat, UCP1 protein reaches up to 5% of
mitochondrial proteins (48), and its thermogenic contribution relies on
its abundance and its activation by free fatty acids. After LPS
stimulation, UCP2 protein increases in lung but reaches only 0.02% of
the mitochondrial proteins, suggesting that either both proteins do not
work the same way or they do not have the same function. On one hand,
since UCP2 and UCP3 are weakly inhibited by purine nucleotides
(49-51), it is possible that UCP2 is constantly activated in
vivo whereas UCP1 is always inhibited by nucleotides in BAT
mitochondria. On the other hand, one could also propose that the
function of the new UCPs is not to trigger thermogenesis by a strong
mitochondrial uncoupling. Negre-Salvayre et al. (14), Lee
et al. (13), and Yang et al. (37) have proposed
that UCP2 regulates the production of reactive oxygen species by
uncoupling the respiratory chain, as suggested by Skulachev (52). In
fact, studies on ucp3(
/
) mice (16) and
ucp2(
/
) mice (17) showed that both proteins do indeed
regulate the production of ROS since higher levels of ROS were observed
in muscle and macrophage, respectively. The finding that UCP2 is
increased in lung and stomach upon LPS treatment and starvation also
support this hypothesis. The kinetics of induction of UCP2 protein
after LPS injection is consistent with a primary immune response
leading to an oxidative burst in lung. Activation of macrophage
receptors by LPS stimulate the production of proinflammatory cytokines
such as TNF
(53) which activates the NF-
B pathway. The cellular
content of reduced glutathione decreases, and consequently, the level
of intracellular ROS increases (for review see Ref. 54). Since
starvation has also been shown to provoke a marked decrease of reduced
glutathione level in lung (54) and in stomach (55), one could propose
that UCP2 does not have any function in basal conditions but is
up-regulated when the level of intracellular reactive oxygen species is
too high. In this context, translational regulation of UCP2 might
ensure a rapid cellular response, based on the large pool of UCP2
mRNA available. Finally, our data point out the lung and the
stomach as two major organs where UCP2 plays an important role. Since
both organs are constantly exposed to toxic compounds and pathogens,
the natural function of UCP2 might be to protect the organism from
oxidative stresses provoked by infection, allergy, or pollution.