|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online April 18, 2008
Journal of Experimental Biology 211, 1456-1462 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012328
Reptilian uncoupling protein: functionality and expression in sub-zero temperatures
1 Laboratoire de Physiologie Intégrative, cellulaire et
moléculaire, Centre National de la Recherche Scientifique (CNRS)
– Université Claude Bernard Lyon 1, 43 Bvd 11 Novembre 1918,
F-69622 Villeurbanne Cedex, France
2 Station d'Ecologie Expérimentale du CNRS à Moulis, Laboratoire
Evolution et Diversité du Vivant, Moulis, F-09200 Saint-Girons,
France
* Author for correspondence at present address: Université Claude Bernard Lyon 1, UMR CNRS 5023 – Ecologie des Hydrosystèmes Fluviaux, Bât. Darwin C, F-69622 Villeurbanne Cedex, France (e-mail: yann.voituron{at}univ-lyon1.fr)
Accepted 3 March 2008
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
coactivator-1
(PGC-1) and peroxisome proliferator-activated receptors (PPAR), mRNA
expression, suggesting that the mechanisms regulating UCP expression may be
conserved between mammals (endotherms) and reptiles (ectotherms). Furthermore,
mitochondria extracted from lizard skeletal muscle showed a guanosine
diphosphate (GDP)-sensitive non phosphorylating respiration. This last result
indicates an inhibition of extra proton leakage mediated by an uncoupling
protein, providing arguments that repUCP is functional in lizard tissues. This
result is associated with a remarkable GDP-dependent increase in mitochondrial
endogenous H2O2 production. All together, these data
support a physiological role of the repUCP in superoxide limitation by lizard
mitochondria in situations of stressful oxidative reperfusion following a
re-warming period in winter.
Key words: Lacerta, cold hardiness, mitochondria, superoxide, supercooling, freezing
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
). The energy devoted to counter this proton leakage
represents roughly 25% of the organism's energy budget, as determined across a
wide range of ectothermic and endothermic vertebrates
(Brand et al., 1991;
Brookes et al., 1998). However,
important differences occur in mitochondrial functioning between endotherms
and ectotherms. Several studies showed (1) a significantly higher
mitochondrial O2 consumption rate in endotherms (5–10 times)
compared to ectotherms (Berner,
1999; Brand et al.,
1991) and (2) that mitochondria from ectotherms are less leaky to
proton than those from endotherms (Brand et
al., 1991; Brookes et al.,
1998). These considerations suggest differences in the fatty acid
composition of mitochondrial membrane phospholipids and/or different degrees
of modulation of mitochondrial proton leakage by proteins such as uncoupling
proteins (UCPs) (Brookes et al.,
1998). UCPs belong to a subfamily of proteins found in the inner
mitochondrial membrane. Until 1997, only one UCP, expressed specifically in
the brown adipose tissue of mammals, was known. However, the discovery of
other proteins sharing about 60% sequence similarity has led to the
classification of supplementary types of UCPs: UCP2 found in various mammalian
tissues and UCP3 mainly expressed in the skeletal muscle
(Ricquier and Bouillaud,
2000). The thermogenic function of mammalian UCP1 is well
established (Cannon and Nedergaard,
2004), but the biological roles of UCP2 and UCP3 are still
controversial. Homologues of mammalian UCP1 have also been detected in a great
variety of organisms such as birds, plants, unicellular organisms and a few
ectothermic vertebrates (Sluse et al.,
2006), suggesting that these proteins have been strongly conserved
through evolution. Despite this probable conservation, evidence of the
presence of UCP in ectothermic vertebrates is scarce and only restricted to
fish (Jastroch et al., 2005;
Liang et al., 2003;
Mark et al., 2006;
Stuart et al., 1999) and
amphibians (Xenopus laevis)
(Keller et al., 2005).
Many hypotheses on the physiological role of UCPs in mammals have been
raised, including the regulation of energy metabolism, the control of body
mass and the attenuation of mitochondrial reactive oxygen species (ROS)
production (Brand and Esteves,
2005; Negre-Salvayre et al.,
1997). In other taxa, the main function of UCP1 homologues also
remains uncertain, however in vitro experiments have shown that they
can modulate the mitochondrial proton motive force, which in turn is a key
factor influencing ROS production at complex I and III of the mitochondrial
respiratory chain (Boveris and Chance,
1973; Papa and Skulachev,
1997). For instance, avian UCP has been shown to protect yeast
mitochondria against the effect of ROS
(Criscuolo et al., 2005).
Furthermore, exogenous superoxide has been reported to activate UCP-mediated
uncoupling in vitro (Echtay et
al., 2002; Krauss et al.,
2003). Therefore, the uncoupling activity of UCPs in vivo
may play a crucial role in protecting tissues from oxidative stress during
periods when overgeneration of ROS is expected
(Papa and Skulachev,
1997).
Low temperatures followed by re-warming are probably one of the most
commonly found ROS-generating stress in nature. Ectotherms survive these
thanks to the contribution of cellular adaptive mechanisms that include,
plasmic and mitochondrial membranes alteration, increase in mitochondrial
volume density, specific isoenzymes and stress proteins synthesis
(Johnston and Bennett, 1996).
At subzero temperatures, freeze tolerance and increasing supercooling
capacities constitute the two means of ectotherm survival. These two
physiological states (frozen and supercooled) induced by sub-zero temperatures
limit oxygen availability to tissues, compelling ectotherms to cope with
potential oxidative stress generated by the transition between ischemic/anoxic
conditions and the reperfusion of oxygenated blood during recovery
(Hermes-Lima and Zenteno-Savin,
2002; Storey,
1996). Both increase in temperature and thawing are then
concomitant with the oxygen reperfusion into tissues restoring organ aerobic
metabolism, and potentially generate oxidative stress. However, data on
reptiles submitted to supercooling, freezing and thawing conditions showed no
significant oxidative damage on DNA and only slight increase of antioxidant
defences (Voituron et al.,
2006). These observations suggest the existence of other adaptive
mechanisms such as ROS limitation within the mitochondria, since mitochondria
are a significant source of ROS in cells, and/or the existence of powerful DNA
repair mechanisms. In the present study, we investigated the potential
existence of a UCP that would reduce ROS production in reptile mitochondria
under subzero temperatures. We report for the first time the detection and the
localization of a UCP homologue in a reptile (Lacerta vivipara). Its
functionality was assessed by measurements of respiration and
H2O2 production of muscle mitochondria. Furthermore, we
also demonstrate the influence of subzero temperatures on its pattern of
expression together with its co-activators peroxisome proliferator-activated
receptor coactivator-1 (PGC-1) and peroxisome
proliferator-activated receptors (PPAR).
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Lacerta vivipara (von Jacquin 1787) individuals, mean body mass 3.27±0.57 g, were captured in late September from a highland population (1450 m) in the Cévennes mountains in France. They were held in boxes with sand and wet moss and cold acclimated at 4°C for 6–7 weeks in the dark with no feeding before being split into four groups. Subsequently, the experiments were performed in November and December.
One group was kept at 4°C as the control group and the remaining lizards where divided among the three groups as described below.
Preparation of supercooled, frozen and thawed animals
Freezing exposures were performed as previously reported
(Voituron et al., 2002a).
Briefly, the lizards in the `frozen' group were placed on a pad of wet
absorbent paper, itself placed in a 50 ml tube. The animals were then
progressively cooled at a constant rate of 0.2°C min–1 in
a low temperature incubator 815 PRECISION (Grenoble, France), from 4°C
(initial temperature) down to the crystallization temperature
(Tc, –2.5°C). We considered that the freezing
exposure of each lizard began immediately after its exotherm and ended when
the individual was removed from its tube 20 h later. During the freezing
period all the individual tubes were placed in an incubation chamber set at
–2.5°C (±0.1°C). A 20-h freezing period was chosen
because it induces an ice content of about 50% and recovery of the frozen
lizards strongly decreased with longer periods of freezing
(Voituron et al., 2002a).
Lizards in the `thawing' group were primarily frozen for 20 h as described for the frozen group but were then thawed in an incubator at +3°C for 24 h.
Lizards in the `supercooling' group were placed on a pad of dry absorbent paper and progressively cooled at a constant rate of 0.2°C min–1 down to –2.5°C. They were then maintained at this temperature for 20 h before being sacrificed.
Lizards from all groups were killed by decapitation and organs were quickly dissected out, frozen in liquid nitrogen and stored at –80°C to await molecular analysis. For the controls and supercooled lizards, blood was collected in heparin, and plasma fatty acid content was determined using the enzymatic NEFA C kit (Wako Chemicals, Neuss, Germany) according to the manufacturer's instructions.
Relative mRNA abundance of repUCP, PGC-1 and PPAR in lizard muscles
Total RNA was extracted from tail muscle samples (80 mg) using Trizol®
(Invitrogen, Cergy Pontoise, France). Concentration and purity were checked by
measuring optimal density at 260 and 280 nm and their integrity was confirmed
by 1% agarose gel electrophoresis (Eurobio, Les Ulis, France). The relative
expression of mRNAs were measured by semi-quantitative reverse transcription
polymerase chain reaction (RT-PCR) using β-actin as standard. For each
sample, a reverse transcription was performed from 1 µg of total RNA with
100 i.u. of M-MLV reverse transcriptase (Promega,
Charbonnières-les-Bains, France), 5 µl of M-MLV RT 5x buffer,
20 i.u. of RNasin ribonuclease inhibitor, 12 pmol of deoxynucleoside
triphosphate and 1 µg of oligo(dT), in a final volume of 25 µl. The
reaction was carried out for 5 min at 70°C [RNA and oligo(dT)], then 60
min at 42°C (all mix) followed by 15 min at 70°C. After chilling, 2.5
µl were used for a PCR reaction. Primer sequences are shown in
Table 1. The 2.5 µl of RT
medium were added to 47.5 µl of PCR mix containing 5 µl 10x REDTaq
PCR buffer, 6 pmol MgCl2, 8 pmol of deoxynucleoside triphosphate,
2.5 i.u. of REDTaq DNA polymerase (Sigma), 22.5 pmoles of corresponding
antisense and sense primers. The PCR conditions were: 2 min at 94°C
followed by 31, 39, 40 or 22 cycles for UCPshort, PPAR, PGC-1
and β-actin, respectively (1 cycle=1 min at 94°C, 1 min at 60°C,
1 min at 72°C). PCR was ended by 10 min at 72°C. Products were
separated on 2% agarose gel prestained with ethidium bromide. For
quantification of relative band intensities, pictures were taken with a DC120
camera (Kodak) and the ratio of each target to β-actin was determined for
each sample with Kodak Digital Science 1D 2.0 (Kodak Scientific Imaging
System, Les Ulis, France).
|
RepUCP expression was also investigated following the same protocol, on a panel of six tissues from control lizards (liver, muscles, lungs, heart, brain and adipose tissue; N=5) to determine if its expression was ubiquitous or tissue specific.
The PCR products obtained with UCPlong primers (Table 1) contained a 780 bp fragment of repUCP and were purified using a Cleamix kit (Talent, Trieste, Italy) and sequenced (Genoscreen, Lille, France).
All the primers used were taken from Gallus gallus sequences (avUCP accession no. AF287144, β-actin accession no. L08165).
Isolation of muscle mitochondria
Leg and tail muscles from four control lizards (4°C for 6–7
weeks) were dissected, pooled and placed in ice-cold isolation buffer (100
mmol l–1 sucrose, 50 mmol l–1 KCl, 5 mmol
l–1 EGTA and 50 mmol l–1 Tris–HCl, pH
7.4). The muscles were chopped with scissors and incubated with 1 mg
g–1 muscle wet weight of protease (Subtilisin A, Sigma, St
Quentin Fallavier, France) for 1 min and then homogenized with a Teflon glass
homogenizer. The mixture was diluted 1:2 (v:v) with isolation buffer without
protease and centrifuged at 800 g for 10 min. The resulting
supernatant was filtered through a cheesecloth and centrifuged at 8000
g for 10 min to obtain the mitochondrial pellet. The pellet
was washed twice with isolation buffer and then centrifuged at 8000
g for 10 min. Finally, the pellet was resuspended in ice-cold
storage medium (250 mmol l–1 sucrose, 20 mmol
l–1 Tris–HCl, 1 mmol l–1 EGTA at pH
7.4). After protein quantification by the Biuret method using BSA as standard,
the mitochondria were diluted to 20 mg ml–1 with storage
medium. All centrifugations were performed at 4°C.
Measurement of mitochondrial O2 consumption
Oxygen consumption was measured with a Clark oxygen electrode (Rank
Brothers Dual Digital, Bioblock, Illkirch, France) in a 0.75 ml glass cell,
thermostatically controlled at 25°C, with constant stirring. The
mitochondria (0.5 mg/ml) were placed in a respiratory medium saturated with
room air, containing 200 mmol l–1 sucrose, 10 mmol
l–1 potassium phosphate and 20 mmol l–1
Tris-HCl, pH 7.4, with 0.3% fatty-acid-free bovine serum albumin (BSA). The
phosphorylating respiration rate (state 3) was measured in the presence of 5
mmol l–1 succinate (current substrate used to study
mitochondrial bioenergetics parameters providing FADH2 to complex
II) and 5 µmol l–1 of rotenone after addition of 1 mmol
l–1 ADP. The control state of respiration (state 4+) was
obtained by the addition of 1 µg/ml oligomycin to inhibit F0-F1 ATPase. The
contribution of UCP to mitochondrial respiration was then assessed by
measuring inhibition of state 4 respiration rate (5 mmol l–1
succinate and 5 µmol l–1 of rotenone) by addition of 2
mmol l–1 guanosine diphosphate (GDP), a known inhibitor of
UCP (Echtay et al., 2002;
Stuart et al., 2001). The
effect of 30 µmol l–1 atractylate on state 4 mitochondrial
respirations was also measured in an independent set of experiments in order
to detect the potential impact of the adenine nucleotide transporter.
Measurement of mitochondrial H2O2 production
The rate of mitochondrial H2O2 release was measured
at 25°C on mitochondria energized with succinate (state 4), following the
linear increase in fluorescence (
ex 312 nm and
em 420 nm) due to oxidation of homovanillic acid (HVA) by
H2O2 in the presence of horseradish (HRP), on a SFM-25
fluorometer (Kontron Instruments, Dardilly, France), as described previously
(Servais et al., 2003).
Reaction conditions were 0.1 mg of mitochondrial protein per ml, HRP (6 i.u.
ml–1), HVA (0.1 mmol l–1) and succinate (5
mmol l–1) as substrates, in the same incubation buffer as was
used for oxygen consumption measurements. Known concentrations of
H2O2 were used to establish a standard concentration
curve. Addition of catalase showed a dose-dependent drop of fluorescence (data
not shown). There was no increase in fluorescence in the absence of substrates
or mitochondria. Measurements of oxygen consumption and
H2O2 release were performed at the same temperature with
the same concentration of substrates in absence or in presence of 1 mmol
l–1 GDP.
Statistical analysis
The data are presented as mean ± s.e.m. The statistical analysis was
performed with the Statview computer statistical package. Data on mRNA
expression (comparing tissues and comparing conditions) was analysed using an
ANOVA followed by a post-hoc Tukey's test, and means values of
mitochondrial respiration were compared using a paired t-test. Blood
fatty acid levels in control and supercooled lizards were compared using a
t-test. A 5% (P<0.05) level of significance was used in
all tests.
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
|
|
, PPAR and repUCP mRNA in lizard muscle and PPAR mRNAs was significantly
increased (P=0.0006 and P=0.01, respectively) with sub-zero
temperatures whatever the physiological states (supercooled state: 1.9- and
2.4-fold, respectively; frozen state: 2.3- and 2.4-fold, respectively)
compared with control lizards (Fig.
3). In lizards thawed for 24 h, the expression of these mRNAs was
still significantly higher than in control lizards (1.7 and 3-fold
respectively), but whereas PPAR reached its highest expression,
PGC-1 expression was significantly lower than its expression under
supercooled and frozen states.
|
O2 consumption of muscle mitochondria from control lizards
We first examined the functioning of the lizard muscle mitochondria using
succinate as substrate (state 4). Addition of ADP to the respiratory medium
leads to oxidative phosphorylation respiration (active state 3=24.4±2.4
natomO min–1 mg–1 protein). Addition of
oligomycin, a specific inhibitor of F0-F1 ATPase, allowed the measurement of
basal O2 consumption (state 4+=14.9± 0.6 natomO
min–1 mg–1 protein), a respiration not
coupled to ATP synthesis. This non-phosphorylating state reflects energy
wastage that is the consequence of proton leakage through the inner
mitochondrial membrane. Proton leakage, which partially uncouples
phosphorylation from oxidation, can be regulated through inducible
UCP-dependent processes (Echtay et al.,
2002; Stuart et al.,
2001; Talbot et al.,
2004). The presence and activity of such a protein in the membrane
of lizard muscle mitochondria were tested using its potent inhibition by
purine nucleoside diphosphates (GDP). Indeed, despite differences between
lower vertebrates and mammals, the response of proton conductance to GDP might
have been conserved during evolution thanks to three specific nucleotides
[Arg83, Arg182 and His214
(Modriansky et al., 1997)]. It
appears that these three nucleotides are conserved in all the UCP isoforms
shown in Fig. 1, providing
arguments that the site of GDP binding is conserved in a number of taxa
including the reptiles.
Fig. 4 and
Table 2 show that state 4
respiration was inhibited by addition of 2 mmol l–1 GDP to
the mitochondrial suspension (–24%), which suggests the presence and the
functionality of an uncoupling protein (repUCP) in lizard muscle mitochondria.
However, a recent study showed that GDP could also interact with the adenine
nucletide transporter (ANT) (Brand et al.,
2005). We therefore tested the effect of an ANT inhibitor (30
µmol l–1 atractylate) and checked that it had no
significant effect on state 4 respiration rate (N=4; paired
t-test, t=–0.64; d.f.=3.1; P>0.05).
Therefore, the reduction of state 4 after addition of GDP presumably results
totally from repUCP inhibition.
|
|
H2O2production of muscle mitochondria from control lizard
The mitochondrion is known to be an important source of ROS production
(Boveris and Chance, 1973;
Nohl, 1994). This production
depends on many factors such as respiratory rate, redox status of the
respiratory complexes (Herrero and Barja,
1998) and the mitochondrial membrane potential
(Papa and Skulachev,
1997).
The rate of H2O2 production by mitochondria, energized with succinate, was clearly dependent upon a reverse electron flow from complex II toward complex I, since it was dramatically inhibited by rotenone (–67%; data not shown). Addition of GDP on state 4 respiration was followed by a significant increase in mitochondrial H2O2 production (+22%; expressed as pmol H2O2 min–1 mg–1 mitochondrial proteins; Table 2). This suggests that inhibition of repUCP by GDP might enhances the mitochondrial membrane potential, amplify the reverse electron flow through complex I and thus increase mitochondrial H2O2 production. As H2O2 production and O2 consumption were measured in the same conditions (buffer, substrate concentration, temperature), we calculated the fraction of O2 turned into H2O2 instead of being reduced to produce water (H2O2 production/O2 consumption), with the result that H2O2 released per unit of oxygen consumed was significantly higher in the presence of GDP (+36%; Table 2), indicating that the uncoupling effect of repUCP contributes to limiting mitochondrial ROS production.
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Even if mRNA levels do not correspond perfectly with protein activity
because of transcriptional regulation
(Pecqueur et al., 2001), the
discovery of a new uncoupling protein in a reptile provides new elements to
the debate regarding the physiological role of these proteins. Indeed, if
initial studies argued for a general function in thermogenesis
(Enerback et al., 1997;
Gong et al., 1997;
Vidal-Puig et al., 2000),
recent work renders the situation less clear, even in mammals
(Harper et al., 2001;
Krauss et al., 2003;
Trenker et al., 2007).
Based on data from literature, we first compared the activation pathway of UCP expression in reptiles and other animals, and secondly considered two possible physiological functions.
FFAs, PGC-1 and PPARs in reptiles at subzero temperatures
Several reports have proposed that, in vivo, fatty acids induce
UCP gene expression in skeletal muscles. In rodents, situations resulting in
high circulating free fatty acid (FFA) levels (such as starvation and cold
exposure) are associated with upregulation of UCP3 mRNA expression
(Schrauwen et al., 2006), but
its expression is low when the FFA levels decrease, as in lactation
(Pedraza et al., 2000).
Accumulating evidence also indicates that fatty acids may act through
peroxisome proliferator-activated receptors (PPAR) to induce UCP gene
expression given that in mammals the UCP promoter contains PPAR-responsive
elements (Solanes et al.,
2003; Son et al.,
2001). Since FFAs are potent ligands for PPAR, it has been
suggested that the upregulation of UCP caused by fasting and other stress
conditions could be explained by increased FFA levels. Once activated by their
respective ligands, PPARs control the transcriptional rate of a large panel of
genes controlling notably lipid and glucose metabolism
(Luquet et al., 2004;
Son et al., 2001).
PPAR activity is regulated by a coactivator (PGC-1; peroxisome
proliferator-activated receptor gamma coactivator-1), originally identified as
a transcriptional coactivator of the nuclear receptor PPAR, which
regulates the activity of several nuclear receptors
(Puigserver and Spiegelman,
2003). PGC-1 mRNA expression is dramatically induced in
both brown fat and skeletal muscle by exposure of animals to cold, and
regulates biogenesis and several key mitochondrial genes, such as UCPs, which
contribute to energy metabolism regulation and the program of adaptive
thermogenesis (Puigserver et al.,
1998). The data presented here, correlating cold-induced increase
of blood FFAs levels (8.0±2.5 and 16.4±4.8 mmol
l–1 in control and supercooled lizards, respectively; data
not shown), PGC-1 and PPAR expression with repUCP mRNA
expression in lizards, are thus totally congruent with other results from the
literature on mammals and suggest a strong conservation through evolution of
this activation pathway.
RepUCP as a fatty acid oxidation inducer?
Since overwintering lizards are exposed to the cold and are aphagic for a
few months, the increased FFA present in the blood and the subsequent PGC-1,
PPAR and repUCP overexpression observed in the present study are clearly a
physiological response to sub-zero temperatures. It is generally acknowledged
that mammalian UCPs play some role in energy metabolism during situations in
which fatty acid oxidation is high. Several studies show a correlation between
overexpression of UCP and stimulation of fatty acid oxidation
(Bezaire et al., 2001). In this
context, the physiological role of UCP might be to export fatty acid anions
from the matrix, thereby preventing the accumulation of fatty acid anions
inside the matrix (Schrauwen et al.,
2001) and protect mitochondria against the detrimental effects of
high fatty acid accumulation (lipotoxicity).
Furthermore, some studies have shown that UCP overexpression in cultured
human muscle cells enhanced fatty acid-dependent inhibition of glucose
oxidation and therefore it has been proposed that UCP could be involved in a
nutrient partitioning process favouring the use of fatty acid over that of
glucose. Moreover, the switching of muscle substrate metabolism to a state of
enhanced lipid utilization during starvation (when glucose is limiting)
suggests UCP could play a role in glucose preservation
(Garcia-Martinez et al.,
2001). In view of these findings and in light of the cold
hardiness framework, we can hypothesize that under sub-zero temperatures
Lacerta vivipara improve their use of lipids as a substrate that will
enable them to preserve glucose for its cryoprotective properties. This
hypothesis is strengthened by the increased level of plasma glucose detected
in overwintering European common lizards during supercooling and freezing
(Voituron et al., 2002a), but
clearly needs further investigation.
RepUCP as mitochondrial free radical regulator?
The increased expression of repUCP can also suggest that these proteins
play an antioxidant role. Indeed, the majority of ROS are generated by the
mitochondrial electron transport chain
(Nohl, 1994;
St-Pierre et al., 2002) and
lead to oxidative stress when an imbalance is observed between the ROS
generation and the detoxification processes. Re-warming tissues after a period
of cold exposure corresponds to a physiological situation comparable with
stressful oxidative situations of tissue ischemia-reperfusion
(Hermes-Lima and Zenteno-Savin,
2002). For instance, freezing exposure stimulates antioxidant
defences against the overproduction of ROS in muscle and lung in the grater
snake (Thamnophis sirtalis) (for a review, see
Storey, 1996). The wood frog
(Rana sylvatica), a model for vertebrate freeze tolerance, displays
considerably higher antioxidant defences in its tissues than the
freeze-intolerant leopard frog (Rana pipiens)
(Storey, 1996). Surprisingly,
data from the literature show low ROS-related damage in frozen–thawed
lizards despite low variations in antioxidant defences
(Voituron et al., 2006). The
possible antioxidant function of the reptilian UCP might thus play significant
role in the biology of reptiles in cold conditions.
Even if the main function of repUCP remains to be established, its
catalytic activity may causes a `mild uncoupling' and may lower ROS generation
by decreasing the reduced state of the mitochondrial respiratory chain as
proposed for mammalian UCP2 and UCP3
(Lambert and Brand, 2004;
Papa and Skulachev, 1997). At
this early point, we demonstrate an inhibition of mitochondrial non
phosphorylating respiration (state 4) by GDP, providing arguments that a mild
uncoupling requiring repUCP occurs in reptile mitochondria and presumably
lowers the proton gradient limiting the ROS generation of mitochondria. This
hypothesis is supported by the fact that inhibition of repUCP by GDP increased
the endogenous mitochondrial production of H2O2.
Towards a `global' cold hardiness strategy in Lacerta vivipara
The two hypotheses proposed above for the role of repUCP (fatty acid
oxidation inducer and antioxidant role) appear complementary rather than
antagonistic. Indeed, Laceta vivipara under sub-zero temperatures
probably (1) preserves glucose for its cryoprotective functions by using fatty
acids as substrate and (2) avoids oxidative damage by preventing generation of
large amounts of ROS.
These data (antioxidant defences and increase in repUCP expression,
especially during supercooling and not freezing) together with those focused
on the aerobic metabolism in both supercooling and freezing states suggest a
`global' strategy in L. vivipara under sub-zero temperatures. Indeed,
previous studies on this species suggested an activation of aerobic metabolic
pathways between 0.5 and –1.5°C
(Voituron et al., 2002b)
allowing the synthesis of different metabolites that probably play a role in
the two cold-hardiness strategies of L. vivipara (freeze tolerance
and freeze avoidance) (Costanzo et al.,
1995). Thus it seems that an adequate cryoprotective system is
activated before the animal reaches its crystallization temperature. The fact
that freezing by itself does induce antioxidant defences
(Voituron et al., 2006)
together with the fact that after 20 h of freezing UCP expression is not
different from that measured in controls, suggests that a freezing without
supercooling will not lead to optimal cryoprotection. If these speculations
are accurate, such a mechanism would have evolved in Lacerta vivipara
in order to cope with the extreme conditions present in their environment, and
needs to be further investigated in future studies.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Berner, N. J. (1999). Oxygen consumption by mitochondria from an endotherm and an ectotherm. Comp. Biochem. Physiol. 124B,25 -31.[CrossRef][Medline]
Bezaire, V., Hofmann, W., Kramer, J. K., Kozak, L. P. and
Harper, M. E. (2001). Effects of fasting on muscle
mitochondrial energetics and fatty acid metabolism in Ucp3(–/–)
and wild-type mice. Am. J. Physiol. Endocrinol. Metab.
281,E975
-E982.
Boveris, A. and Chance, B. (1973). The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem. J. 134,707 -716.[Medline]
Brand, M. D. and Esteves, T. C. (2005). Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3. Cell Metab. 2,85 -93.[CrossRef][Medline]
Brand, M. D., Couture, P., Else, P. L., Withers, K. W. and Hulbert, A. J. (1991). Evolution of energy metabolism. Proton permeability of the inner membrane of liver mitochondria is greater in a mammal than in a reptile. Biochem. J. 275, 81-86.[Medline]
Brand, M. D., Pakay, J. L., Ocloo, A., Kokoszka, J., Wallace, D. C., Brookes, P. S. and Cornwall, E. J. (2005). The basal proton conductance of mitochondria depends on adenine nucleotide translocase content. Biochem. J. 392,353 -362.[CrossRef][Medline]
Brookes, P. S., Buckingham, J. A., Tenreiro, A. M., Hulbert, A. J. and Brand, M. D. (1998). The proton permeability of the inner membrane of liver mitochondria from ectothermic and endothermic vertebrates and from obese rats: correlations with standard metabolic rate and phospholipid fatty acid composition. Comp. Biochem. Physiol. 119B,325 -334.[CrossRef][Medline]
Cannon, B. and Nedergaard, J. (2004). Brown
adipose tissue: function and physiological significance. Physiol.
Rev. 84,277
-359.
Costanzo, J. P., Grenot, C. and Lee, R. E. (1995). Supercooling, ice nucleation and freeze tolerance in the european common lizard, Lacerta vivipara. J. Comp. Physiol. B 165,238 -244.[Medline]
Criscuolo, F., Gonzalez-Barroso Mdel, M., Le Maho, Y., Ricquier,
D. and Bouillaud, F. (2005). Avian uncoupling protein
expressed in yeast mitochondria prevents endogenous free radical damage.
Proc. Biol. Sci. 272,803
-810.
Echtay, K. S., Roussel, D., St-Pierre, J., Jekabsons, M. B., Cadenas, S., Stuart, J. A., Harper, J. A., Roebuck, S. J., Morrison, A., Pickering, S. et al. (2002). Superoxide activates mitochondrial uncoupling proteins. Nature 415, 96-99.[CrossRef][Medline]
Enerback, S., Jacobsson, A., Simpson, E. M., Guerra, C., Yamashita, H., Harper, M. E. and Kozak, L. P. (1997). Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 387,90 -94.[CrossRef][Medline]
Garcia-Martinez, C., Sibille, B., Solanes, G., Darimont, C.,
Mace, K., Villarroya, F. and Gomez-Foix, A. M. (2001).
Overexpression of UCP3 in cultured human muscle lowers mitochondrial membrane
potential, raises ATP/ADP ratio, and favors fatty acid vs. glucose oxidation.
FASEB J. 15,2033
-2035.
Gong, D. W., He, Y., Karas, M. and Reitman, M.
(1997). Uncoupling protein-3 is a mediator of thermogenesis
regulated by thyroid hormone, beta3-adrenergic agonists, and leptin.
J. Biol. Chem. 272,24129
-24132.
Harper, M. E., Dent, R. M., Bezaire, V., Antoniou, A., Gauthier, A., Monemdjou, S. and McPherson, R. (2001). UCP3 and its putative function: consistencies and controversies. Biochem. Soc. Trans. 29,768 -773.[CrossRef][Medline]
Hermes-Lima, M. and Zenteno-Savin, T. (2002). Animal response to drastic changes in oxygen availability and physiological oxidative stress. Comp. Biochem. Physiol. 133C,537 -556.[CrossRef][Medline]
Herrero, A. and Barja, G. (1998). H2O2 production of heart mitochondria and aging rate are slower in canaries and parakeets than in mice: sites of free radical generation and mechanisms involved. Mech. Ageing Dev. 103,133 -146.[CrossRef][Medline]
Jastroch, M., Wuertz, S., Kloas, W. and Klingenspor, M.
(2005). Uncoupling protein 1 in fish uncovers an ancient
evolutionary history of mammalian nonshivering thermogenesis.
Physiol. Genomics 22,150
-156.
Johnston, I. A. and Bennett, A. F. (ed.) (1996). Animals and Temperature: Phenotypic and Evolutionary Adaptation (Society for Experimental Biology Seminar Series 59). Cambridge: Cambridge University Press.
Keller, P. A., Lehr, L., Giacobino, J. P., Charnay, Y.,
Assimacopoulos-Jeannet, F. and Giovannini, N. (2005).
Cloning, ontogenesis, and localization of an atypical uncoupling protein 4 in
Xenopus laevis. Physiol. Genomics
22,339
-345.
Krauss, S., Zhang, C. Y., Scorrano, L., Dalgaard, L. T., St-Pierre, J., Grey, S. T. and Lowell, B. B. (2003). Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction. J. Clin. Invest. 112,1831 -1842.[CrossRef][Medline]
Lambert, A. J. and Brand, M. D. (2004). Superoxide production by NADH:ubiquinone oxidoreductase (complex I) depends on the pH gradient across the mitochondrial inner membrane. Biochem. J. 382,511 -517.[CrossRef][Medline]
Liang, X. F., Ogata, H. Y., Oku, H., Chen, J. and Hwang, F. (2003). Abundant and constant expression of uncoupling protein 2 in the liver of red sea bream Pagrus major. Comp. Biochem. Physiol. 136A,655 -661.
Luquet, S., Lopez-Soriano, J., Holst, D., Gaudel, C., Jehl-Pietri, C., Fredenrich, A. and Grimaldi, P. A. (2004). Roles of peroxisome proliferator-activated receptor delta (PPARdelta) in the control of fatty acid catabolism. A new target for the treatment of metabolic syndrome. Biochimie 86,833 -837.[Medline]
Mark, F. C., Lucassen, M. and Pörtner, H. O. (2006). Thermal sensitivity of uncoupling protein expression in polar and temperate fish. Comp. Biochem. Physiol. 1D,365 -374.
Modriansky, M., Murdza-Inglis, D. L., Patel, H. V., Freeman, K.
B. and Garlid, K. D. (1997). Identification by site-directed
mutagenesis of three arginines in uncoupling protein that are essential for
nucleotide binding and inhibition. J. Biol. Chem.
272,24759
-24762.
Negre-Salvayre, A., Hirtz, C., Carrera, G., Cazenave, R., Troly, M., Salvayre, R., Penicaud, L. and Casteilla, L. (1997). A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J. 11,809 -815.[Abstract]
Nobes, C. D., Brown, G. C., Olive, P. N. and Brand, M. D.
(1990). Non-ohmic proton conductance of the mitochondrial inner
membrane in hepatocytes. J. Biol. Chem.
265,12903
-12909.
Nohl, H. (1994). Generation of superoxide radicals as byproduct of cellular respiration. Ann. Biol. Clin. Paris 52,199 -204.[Medline]
Papa, S. and Skulachev, V. P. (1997). Reactive oxygen species, mitochondria, apoptosis and aging. Mol. Cell. Biochem. 174,305 -319.[CrossRef][Medline]
Pecqueur, C., Alves-Guerra, M. C., Gelly, C., Levi-Meyrueis, C.,
Couplan, E., Collins, S., Ricquier, D., Bouillaud, F. and Miroux, B.
(2001). Uncoupling protein 2, in vivo distribution,
induction upon oxidative stress, and evidence for translational regulation.
J. Biol. Chem. 276,8705
-8712.
Pedraza, N., Solanes, G., Carmona, M. C., Iglesias, R., Vinas, O., Mampel, T., Vazquez, M., Giralt, M. and Villarroya, F. (2000). Impaired expression of the uncoupling protein-3 gene in skeletal muscle during lactation: fibrates and troglitazone reverse lactation-induced downregulation of the uncoupling protein-3 gene. Diabetes 49,1224 -1230.[Abstract]
Puigserver, P. and Spiegelman, B. M. (2003).
Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1
alpha): transcriptional coactivator and metabolic regulator.
Endocr. Rev. 24,78
-90.
Puigserver, P., Wu, Z., Park, C. W., Graves, R., Wright, M. and Spiegelman, B. M. (1998). A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92,829 -839.[CrossRef][Medline]
Ricquier, D. and Bouillaud, F. (2000). The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem. J. 345,161 -179.[CrossRef][Medline]
Schrauwen, P., Saris, W. H. and Hesselink, M. K.
(2001). An alternative function for human uncoupling protein 3,
protection of mitochondria against accumulation of nonesterified fatty acids
inside the mitochondrial matrix. FASEB J.
15,2497
-2502.
Schrauwen, P., Hoeks, J. and Hesselink, M. K. (2006). Putative function and physiological relevance of the mitochondrial uncoupling protein-3: involvement in fatty acid metabolism? Prog. Lipid Res. 45,17 -41.[CrossRef][Medline]
Servais, S., Couturier, K., Koubi, H., Rouanet, J. L., Desplanches, D., Sornay-Mayet, M. H., Sempore, B., Lavoie, J. M. and Favier, R. (2003). Effect of voluntary exercise on H2O2 release by subsarcolemmal and intermyofibrillar mitochondria. Free Radic. Biol. Med. 35, 24-32.[CrossRef][Medline]
Sluse, F. E., Jarmuszkiewicz, W., Navet, R., Douette, P., Mathy, G. and Sluse-Goffart, C. M. (2006). Mitochondrial UCPs: new insights into regulation and impact. Biochim. Biophys. Acta 1757,480 -485.[Medline]
Solanes, G., Pedraza, N., Iglesias, R., Giralt, M. and
Villarroya, F. (2003). Functional relationship between MyoD
and peroxisome proliferator-activated receptor-dependent regulatory pathways
in the control of the human uncoupling protein-3 gene transcription.
Mol. Endocrinol. 17,1944
-1958.
Son, C., Hosoda, K., Matsuda, J., Fujikura, J., Yonemitsu, S.,
Iwakura, H., Masuzaki, H., Ogawa, Y., Hayashi, T., Itoh, H. et al.
(2001). Up-regulation of uncoupling protein 3 gene expression by
fatty acids and agonists for PPARs in L6 myotubes.
Endocrinology 142,4189
-4194.
Storey, K. B. (1996). Oxidative stress: animal adaptations in nature. Braz. J. Med. Biol. Res. 29,1715 -1733.[Medline]
St-Pierre, J., Buckingham, J. A., Roebuck, S. J. and Brand, M.
D. (2002). Topology of superoxide production from different
sites in the mitochondrial electron transport chain. J. Biol.
Chem. 277,44784
-44790.
Stuart, J. A., Harper, J. A., Brindle, K. M. and Brand, M. D. (1999). Uncoupling protein 2 from carp and zebrafish, ectothermic vertebrates. Biochim. Biophys. Acta 1413,50 -54.[Medline]
Stuart, J. A., Harper, J. A., Brindle, K. M., Jekabsons, M. B. and Brand, M. D. (2001). A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast. Biochem. J. 356,779 -789.[CrossRef][Medline]
Talbot, D. A., Lambert, A. J. and Brand, M. D. (2004). Production of endogenous matrix superoxide from mitochondrial complex I leads to activation of uncoupling protein 3. FEBS Lett. 556,111 -115.[CrossRef][Medline]
Trenker, M., Malli, R., Fertschai, I., Levak-Frank, S. and Graier, W. F. (2007). Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport. Nat. Cell Biol. 9,445 -452.[CrossRef][Medline]
Vidal-Puig, A. J., Grujic, D., Zhang, C. Y., Hagen, T., Boss,
O., Ido, Y., Szczepanik, A., Wade, J., Mootha, V., Cortright, R. et al.
(2000). Energy metabolism in uncoupling protein 3 gene knockout
mice. J. Biol. Chem.
275,16258
-16266.
Voituron, Y., Storey, J. M., Grenot, C. and Storey, K. B. (2002a). Freezing survival, body ice content and blood composition of the freeze tolerant European common lizard, Lacerta vivipara. J. Comp. Physiol. B 172, 71-76.[CrossRef][Medline]
Voituron, Y., Verdier, B. and Grenot, C. (2002b). The respiratory metabolism of a lizard (Lacerta vivipara) in supercooled and frozen states. Am. J. Physiol. 283,R181 -R186.
Voituron, Y., Servais, S., Romestaing, C., Douki, T. and Barré, H. (2006). Oxidative DNA damage and antioxidant defenses in the European common lizard (Lacerta vivipara) in supercooled and frozen states. Cryobiology 52, 74-82.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
