Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish.

Exploring Uncoupling Proteins and Antioxidant
Mechanisms under Acute Cold Exposure in Brains of Fish
Yung-Che Tseng1,2., Ruo-Dong Chen1,3., Magnus Lucassen4, Maike M. Schmidt5, Ralf Dringen5, Doris
Abele4, Pung-Pung Hwang1*
1 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei City, Taiwan, 2 Leibniz-Institute of Marine Sciences, IFM-GEOMAR, Biological Oceanography, Kiel,
Germany, 3 Institute of Zoology, National Taiwan University, Taipei City, Taiwan, 4Alfred-Wegener Institute for Polar and Marine Research, Bremerhaven, Germany,
5Center for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany
Abstract
Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of
mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential
oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study,
the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure
(from 28uC to 18uC) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA
level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%–90% (1 and 24 h), respectively,
while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a
very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the
mRNA level of the glucose transporter zglut3 in brain by 50%–100%. To test for an involvement of uncoupling proteins
(UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of
zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels
of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold
exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure
to 18uC. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic
balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure.
Citation: Tseng Y-C, Chen R-D, Lucassen M, Schmidt MM, Dringen R, et al. (2011) Exploring Uncoupling Proteins and Antioxidant Mechanisms under Acute Cold
Exposure in Brains of Fish. PLoS ONE 6(3): e18180. doi:10.1371/journal.pone.0018180
Editor: Michael Polymenis, Texas A&M University, United States of America
Received November 26, 2010; Accepted February 22, 2011; Published March 25, 2011
Copyright: � 2011 Tseng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was financially supported by Academia Sinica (P. P. Hwang) and Alfred-Wegener Institute for Polar and Marine Research (D. Abele). Y. C.
Tseng was awarded the Postdoc Scholarship supported by both the National Science of Council of Taiwan and the Bilateral Cooperation in Science and
Technology grant from International Bureau of the Germany Federal Ministry of Education and Research. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: pphwang@gate.sinica.edu.tw
. These authors contributed equally to this work.
Introduction
The vertebrate brain may be the organ most vulnerable to
thermal fluctuations, since most of the physiological acclimation
responses are initiated by the central nervous system (CNS) [1].
Cold shock causes severe pathologies in the mammalian brain [2],
whereas mild cooling (to 32uC) can support survival and delay the
deleterious effects of infarction [3]. Contrary, ectothermic verte-
brates regularly experience brain cooling during day/night cycles of
thermal fluctuations, or on seasonal scales, during winter in
temperate regions. An early study on green sunfish (Lepomis cyanellus)
demonstrates elevated activities of glycolytic enzymes, such as
glucosephosphate isomerase, glyceraldehydephosphate dehydroge-
nase, and pyruvate kinase, in the brain during cold exposure [4].
The high metabolic rate of brain cells implies a high production of
ROS [5]. In addition, homeoviscous adaptations, i.e. increased
polyunsaturation of membrane phospholipids that maintain
membrane fluidity in the brain during prolonged exposure to cold
temperature [6,7] enhance the susceptibility to oxidative stress [8].
Malek et al. [9] found a suite of antioxidant enzymes, including
several superoxide dismutase (SOD) and glutathione peroxidase
(GPx) isoforms and thioredoxin, but not catalase (CAT), up-
regulated in zebrafish skeletal muscle, following temperature
reduction from 28 to 18uC within 4 weeks and subsequent 6
months of maintenance at 18uC. In spite of the molecular
antioxidant response, oxidative stress markers of lipid and protein
oxidation and 8-oxodG-DNA damage were slightly higher in cold
exposed zebrafish muscle. Thus, oxidative stress is indeed an issue
during prolonged cold exposure in fish, which may also relate to the
slow down of cellular repair mechanisms in the cold [10].
To avoid oxidative stress and keep cellular redox state in
balance, aerobic cells utilize small molecular weight antioxidants
and antioxidative enzymes. By activation of enzymes such as
SOD, CAT, and peroxidases, cells respond to acute challenges.
Low molecular weight antioxidants, such as ascorbate, glutathione
(GSH), and phenolic compounds, contribute to the basal ROS
scavenging antioxidant protection [11], often depending also on
life history or feeding state.
Furthermore, ‘‘mild uncoupling’’ of the mitochondrial inner
membrane controls the membrane potential and limits mito-
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chondrial ROS production [12,13,14]. Oxidative phosphoryla-
tion is never fully coupled to ATP synthesis in vivo, conditional
mainly on the existence of uncoupling proteins (UCPs) [15].
UCPs belong to a superfamily of mitochondrial anion-carriers
(SLC25A) located in the mitochondrial inner membrane with a
molecular mass of 31–34 kDa [16]. UCPs catalyze proton
conductance and dissipate the mitochondrial gradient required
for ATP production, which results in a reduction of mitochon-
drial membrane potential (DYm) and mitigates ROS formation,
especially at respiratory complex I [17]. UCP activity is under
close control of different effectors and inhibitors: it is strongly
inhibited by purine nucleotides such as ATP and GDP and, in
turn, activated by free-fatty acids and superoxide [18].
Moreover, the lipid peroxidation product 4-hydroxynoneal
(HNE), also activates UCPs, and may serve as mediator of mild
uncoupling, especially when mitochondrial membrane potential
is high [12].
Five mammalian UCP homologues, UCP1-UCP5 are known.
The original UCP, UCP1 (SLC25A7, thermogenin, 10% of
membrane bound protein) is well known for its role in adaptive
non-shivering thermogenesis (NST) in brown adipose tissue [19].
UCP2 is expressed in various tissues including brain, while UCP3
is expressed mainly in heart and skeletal muscles [20]. UCP4
(SLC25A27) and UCP5 (SLC25A14, also called BMCP1) are both
expressed mainly in the nervous system, however, their functional
characteristics are not well studied and their physiological roles are
still unknown [15,21]. The discovery of UCP homologues in
ectotherms raises the question concerning their primary physio-
logical role in thermoregulation [22,23,24,25,26]. On one hand,
some piecemeal fish UCP paralogs have already been cloned
[22,24,25]; on the other hand, in crocodile and several species of
fish, mRNA levels or protein expressions of UCP homologues
change in response to acute or seasonal temperature variations.
Furthermore, the UCP genomic studies in fish and invertebrates
raised an adaptive evolution scenario with respect to non-shivering
thermogenesis (NST). Gene duplication and loss occurred in some
lineages which was closely related to the UCPs’ functional diversity
between eutherians, the mammalian ancestors and ectotherms
[27,28].
PPARs (peroxisome proliferator-activated receptors) are ligand-
activated transcription factors which belong to the nuclear
receptor superfamily and regulate the expression of target genes
involved in lipid and energy metabolism [29]. PPARs have been
identified as pivotal actors in the control of UCPs gene
transcription [30] in their respective tissues [31,32]. In addition,
PPARa also activates the expression of antioxidant enzymes,
including SOD and GPx [33]. To date, three PPAR isoforms have
been characterized in mammals: PPARa PPARb/d, and PPARc,
each isoform with a unique expression pattern relating to their
distinct cellular functions [30].
Cold-induced oxidative stress has been suggested to play key
role in brain damage. Neuronal UCPs are induced by oxidative
stress products and by superoxide and seem to be crucial for
reducing the mitochondrial ROS production [34]. The present
paper is aimed at further investigating the neuroprotective
effects of UCPs in fish brain, especially with respect to how
UCPs are controlled under cold-induced oxidative stress in the
fish CNS. The physiological role of PPARs in UCP gene
expression and the mechanism of PPARs in the prevention of
oxidative stress and neuroprotection have been reported in
mammals [30,31,32,33]. One of these studies suggests that
UCPs may be involved in PPAR dependent gene transactivation
through intrachromosomal looping next to their uncoupling
function in the mitochondria [31]. Another important tran-
scription factor involved in temperature control of gene
transcription, although via an indirect effect of temperature
induced hypoxia, is the hypoxia inducible factor HIF-1. HIF-1
protein stabilization was observed in temperate eelpout (Zoarces
viviparus) during winter cold [35,36]. Parallel UCP2 was up-
regulated in cold adapted eelpout [25]. In addition, ROS over
production in CNS caused the expressions of HIF-1 responsive
genes, such as glucose transporters (GLUTs), vascular endo-
thelial growth factor (VEGF), and erythropoietin (EPO), for
supporting ATP production and facilitating oxygen supply [37].
There is considerable evidence supporting the issue of
bidirectional crosstalk between mitochondrial ROS and HIF
activity. These ROS may act as signaling molecules that
somehow influence the regulation of the HIF pathway during
hypoxia [38,39,40].
Although the response to cold challenge in teleost fish has been
intensively studied, the molecular and physiological mechanisms
and mutual relations protecting fish CNS against cold induced
ROS damage are not at all understood. Therefore, particular
attention should be paid on the regulatory aspects of gene
transcription, involving HIF and the PPAR/UCP system.
Moreover, the CNS cellular metabolism modulation should also
be examined. In the present study we used the warm adapted
zebrafish model, Danio rerio, to study the effects of acute cold
exposure (from 28uC to 18uC) on fish brain. The zebrafish model
is backed by a genetic database, and its applicability to study
various molecular/cellular pathways and pathologies has been
confirmed [41,42]. UCP homologs in zebrafish were explored
from genomic sequence analysis to transcript expressions.
Specifically, we have, for the first time, measured the expression
levels of UCP/PPAR, an oxidative stress parameter and several
antioxidative parameters in the CNS of zebrafish upon acute cold
exposure. In addition, HIF-1a protein content and the transcript
levels of HIF-regulated GLUTs were quantified to test for an
involvement of UCPs and HIF in modulating stress during cold
exposure in fish brain.
Results
Phylogenetic analysis, sequence identity and gene
structures of zUCPs
Multiple sequence alignment and phylogenetic (NJ) analysis
with homologues of other species clearly identified 5 members
of the zUCP family which enables unambiguous identification
of the zebrafish homologues (Fig. 1). The relative sequence
identity between zUCPs is shown in Table 1. To further identify
these zucp genes, comprehensive searches were performed to
confirm these orthologs and determine their genomic locations
(Figs. 2, 3, 4, 5). In the genome sequences of zebrafish, each
isoform of zucps was located on different chromosome, except
zucp2 and zucp2l, which were located between 34.13 mega base-
pairs (Mb) and 34.21 Mb on the same chromosome 10 (Fig. 3).
Further, gene arrangements in the genomic regions encompass-
ing ucps were compared. As shown in Figs. 2, 3, 4, 5, zucp genes
have their own syntenies, meaning that they co-localize adjacent
to different genes than in mammals and higher ectotherms, but
the genetic environment also differs in the pufferfish. Compar-
ing the gene arrangements between each zucp isoform (Figs. 2, 3,
4, 5), conserved synteny of ucp1 was found across humans,
rodents, amphibians (Fig. 2). In the genome sequences of human
and rodent ucp2 and ucp3 are located next to each other,
whereas chicken and amphibians have either ucp3 or ucp2,
respectively (Fig. 3). Exact phylogenetic analysis, revealed ucp2-
like (ucp2l) genes are newly annotated and only found in fish
UCPs and Oxidative Stress in Zebrafish Brain
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(Fig. 3). In addition, gene arrangements of ucp2 and ucp2l in the
genomic regions encompassing these fish isoforms were
compared. These homologues in zebrafish, tetradon and
pufferfish are located adjacent to ucp2 on the same chromosome
(Fig. 3). Phylogenetic inference grouped SLC25A27 (UCP4) and
SLC25A14 (UCP5) into another root from other members
(Fig. 1). The syntenies found around ucp4 and ucp5 between
humans and rodents are not conserved across chicken,
amphibians, zebrafish, tetraodon (Tetraodon nigroviridis), and
pufferfish (Takifugu rubripes) (Figs. 4, 5).
zucp mRNA expressions in zebrafish at 28uC
Expression of zucp mRNAs was evaluated by RT-PCR in
different zebrafish tissues (Fig. 6). All isoforms were expressed in
brain, heart, spleen, intestinal wall and kidney. mRNA expressions
of the zucp2 and zucp5 isoforms were ubiquitously expressed in all
Figure 1. Phylogenetic analysis of UCP amino acid sequences. The putative sequences of other species and zebrafish (Danio rerio) were
obtained from the NCBI and Ensembl databases as listed in Supporting Information S2. Consensus trees were generated using the Neighbor-joining
method with the pairwise deletion gap calculating option. The results were confirmed by 1,000 bootstraps. The unit of scale bar is the number of
amino acid substitutions per site.
doi:10.1371/journal.pone.0018180.g001
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tissues. Contrary, mRNA levels of zucp4 were low compared to
other isoforms in all tissues.
Localization of zucp mRNAs in zebrafish brain at 28uC
In subsequent experiments, specific RNA probes were
designed to conduct in situ hybridization of the 5 zucp isoforms
in different horizontal axis cryo-sections of zebrafish brain. As
shown in Fig. 7A, zucp1 was predominately localized in the
anterior part of the medial division of the cerebellar crest (CC),
the valvula crebelli (Vam), the parvocellular preoptic nucleus
(PPa), the periventricular gray zone of the optic tectum (PGZ)
and the ventromedial thalamic nucleus (VM). mRNA of zucp2
was strongly stained in CC, caudal lobe of the cerebellum
(LCa), the cerebellar corpus (CCe), the dorsal posterior
thalamic nucleus (DP), the granular eminence (EG), the
habenula (Ha), the lateral nucleus of the ventral telecephalic
area (Vl), the longitudinal torus (TL), the lateral division of the
valvula cerebelli (Val), PGZ and VM (Fig. 7B). Positive signals
for zucp2l were observed in the central posterior thalamic
nucleus (CP), CC, PGZ, Vam and VM (Fig. 7C). Further,
expressions of zucp4 in zebrafish brains were found in CC,
CCe, EG, LCa, PGZ, periventricular nucleus of posterior
tuberculum (TPp), Val and VM (Fig. 7D). Furthermore, zucp5
was localized in brain areas of CCe, EG, LCa, PGZ and VM
(Fig. 7E). In situ hybrization indicated all zucp homologues to be
expressed in PGZ and VM regions under control conditions
(28uC).
Effects of cold acclimation on mRNA expressions patterns
of zucps in zebrafish brain
The time-course changes of zucps’ mRNA expression in brain
of zebrafish transferred from 28uC to 18uC at different times of
cooling up to 24 h are shown in Fig. 8, with ribosomal protein
L13a (zrpl13a) as house keeping gene. At 28uC control
temperature, zucp1 and zucp5 were stronger expressed in brains
compared to zucp2l and zucp4 (Fig. 8). Furthermore, zucp5
exhibited the highest mRNA levels in brains of zebrafish at
control temperature and was about 80-fold higher expressed
than zucp4. During cold-shock (1 h after transfer) and a 6 and
24 h of cold acclimation, zucp1 transcript expression remained
invariably high without any change, whereas expression of
zucp2 increased significantly by about 3-fold at 6 and 24 h of
cold exposure compared to control group. In addition, the
zucp2l mRNA was significantly induced after 24 h at 18uC.
Expression of zucp4 transcript in brain was always very low, but
was rapidly up-regulated within 1 h of acute cold shock,
whereafter expression returned to control level at 6 h cooling at
Figure 2. Gene structures encompassing UCP1 orthologs. The physical distance of the genomic region is indicated on both sides. Chr., the
chromosome. The arrow indicates the gene with the direction. All sequences of UCP orthologs obtained from the NCBI and Ensembl database are
referring to Supporting Information S2. The un-annotated proteins of Xenopus tropicalis and pufferfish were obtained from the Ensembl database.
ENSXETT and ENSTRUT indicate the symbols of Ensembl transcript ID of Xenopus tropicalis and pufferfish, respectively. Those zucp neighboring
transcripts were identified utilizing the Ensembl genome browser system. ELMOD2, ELMO/CED-12 domain containing 2; INPP4B, inositol
polyphosphate-4-phosphatase, type II; TBC1D9, TBC1 domain family, member 9.
doi:10.1371/journal.pone.0018180.g002
Table 1. Identities (in percent) of amino acid sequences
among the identified zebrafish UCP (zUCP) isoforms.
Identity
(%) zucp1 zucp2 zucp2l zucp4 zucp5
zucp1 - 72 46 33 33
zucp2 - 50 32 34
zucp2l - 23 22
zucp4 - 36
zucp5 -
doi:10.1371/journal.pone.0018180.t001
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18uC. After 24 h at 18uC, zucp4 transcript levels were lower
than in control fish brain at 28uC. The time profile of brain
zucp5 mRNA expression resembled that of zucp2l. However, the
expression level was generally higher than zucp2l and up-
regulation of zucp5 transcript 1 h after transfer to 18uC was
significant. At 6 h of cold exposure the expression declined to
below control level (28uC), but recovered again reaching
control and 1 h cold exposure levels at 24 h of cold exposure.
Figure 4. Gene structures encompassing UCP4 orthologs. The physical distance of the genomic region is indicated on both sides. Chr., the
chromosome. The arrow indicates the gene with the direction. All sequences of UCP orthologs obtained from the NCBI and Ensembl database are
referring to Supporting Information S2. The un-annotated protein of Xenopus tropicalis was obtained from the Ensembl database. ENSXETT indicates
the symbols of Ensembl transcript ID of Xenopus tropicalis. Those zucp neighboring transcripts were identified utilizing the Ensembl genome browser
system. C6orf115, chromosome 6 open reading frame 115; CYP39A1, cytochrome P450, family 39, subfamily A, polypeptide 1; si:dkey-34f16.5, si:dkey-
34f16.5 protein; TDRD6, tudor domain containing 6.
doi:10.1371/journal.pone.0018180.g004
Figure 3. Gene structures encompassing UCP2 and UCP3 orthologs. The physical distance of the genomic region is indicated on both sides.
Chr., the chromosome. The arrow indicates the gene with the direction. All sequences of UCP orthologs obtained from the NCBI and Ensembl
database are referring to Supporting Information S2. The un-annotated proteins of tetraodon, and pufferfish were obtained from the Ensembl
database. GSTENT and ENSTRUT indicate the symbols of Ensembl transcript ID of tetraodon, and pufferfish, respectively. Those zucp neighboring
transcripts were identified utilizing the Ensembl genome browser system. C2CD3, C2 calcium-dependent domain containing 3; DNAJB13, DnaJ
(Hsp40) homolog, subfamily B, member 13; PPME1, protein phosphatase methylesterase 1; si:dkey-21n12.1, si:dkey-21n12.1 protein.
doi:10.1371/journal.pone.0018180.g003
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