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Physiol. Genomics 17: 130-139, 2004. First published February 17, 2004; doi:10.1152/physiolgenomics.00165.2003
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Received 26 September 2003; accepted in final form 13 February 2004.
Physiological Genomics 17:130-139 (2004)
1094-8341/04 $5.00 © 2004 American Physiological Society

Uncoupling protein 2 and 3 in marsupials: identification, phylogeny, and gene expression in response to cold and fasting in Antechinus flavipes

Martin Jastroch1, Kerry Withers2 and Martin Klingenspor1

1 Animal Physiology, Department of Biology, Philipps-University Marburg, 35032 Marburg, Germany
2 Department of Biological and Physical Sciences, University of Southern Queensland, Toowoomba, Queensland 4350, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We searched for the presence of uncoupling protein genes so far unknown in marsupials and monotremes and identified uncoupling protein 2 (UCP2) and UCP3 full-length cDNAs in libraries constructed from the marsupials Antechinus flavipes and Sminthopsis macroura. Marsupial UCP2 is 89–90% identical to rodent UCP2, whereas UCP3 exhibits 80% identity to mouse UCP3. A phylogenetic tree including all known UCPs positions the novel marsupial UCP2 and UCP3 at the base of the mammalian orthologs. In the 5'-untranslated region of UCP2 a second open reading frame encoding for a 36-amino acid peptide was identified which is highly conserved in all vertebrate UCP2 transcripts. Analysis of tissue specificity in A. flavipes with homologous cDNA probes revealed ubiquitous presence of UCP2 mRNA and striated muscle specificity of UCP3 mRNA resembling the known expression pattern in rodents. Neither UCP2 nor UCP3 gene expression was stimulated in adipose tissue and skeletal muscle of cold exposed A. flavipes. However, UCP3 mRNA expression was upregulated 6-fold in heart and 2.5-fold in skeletal muscle as reported for rodents in response to fasting. Furthermore, UCP3 mRNA seems to be coregulated with PDK4 mRNA, indicating a relation to enhanced lipid metabolism. In contrast, UCP2 gene expression was not regulated in response to fasting in adipose tissue and skeletal muscle but was diminished in the lung and increased in adipose tissue. Taken together, the sequence analysis, tissue specificity and physiological regulation suggest a conserved function of UCP2 and UCP3 during 130 million years of mammalian evolution.

Sminthopsis macroura; Tachyglossus aculeatus; pyruvate dehydrogenase 4; monotremes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IN EUTHERIAN MAMMALS, three uncoupling proteins (UCPs) related to the mitochondrial anion carrier family have been identified (3). The thermogenic UCP1 uncouples the respiratory chain from ATP synthesis in brown adipose tissue (BAT) mitochondria (41), but the function of more recently discovered UCPs has not been resolved (40). While UCP1 and UCP3 were so far only described in placental mammals, UCP2 was also found only in fish (58). Other members of the UCP family have been identified in plants (35).

Although some UCPs show rather distinct tissue-specific expression patterns, e.g., UCP1 in BAT only and UCP3 in BAT, heart, and skeletal muscle, UCP2 mRNA is present at variable levels in multiple tissues of eutherian mammals (5, 20, 37). The uncoupling activity of UCP1, UCP2, and UCP3 is increased by superoxides, suggesting a more general role of these mitochondrial anion carriers in detoxification of reactive oxygen species (ROS) (15). In contrast, other reports failed to demonstrate superoxide-induced uncoupling of UCP2 (11). The physiological function of UCPs other than UCP1 has not been finally resolved yet, and there exists considerable doubt as to whether UCP2 and UCP3 are indeed uncouplers of mitochondrial respiration (40). Alternative hypotheses on the putative function of UCP2 and UCP3 have been suggested and are currently under investigation. UCP3 is a potential modulator of fatty acid oxidation in mitochondria (24), whereas UCP2 may be involved in inflammatory responses and/or the regulation of insulin secretion from pancreatic ß-cells (1, 18, 20, 29, 64). In rodents, the expression of UCP3 mRNA is induced in BAT but not in skeletal muscle after 48 h of cold exposure, whereas in response to fasting expression is only increased in skeletal muscle (7, 59). The highest UCP2 mRNA expression is found in tissues rich with macrophages and can be regulated in these cells by injection of lipopolysaccharides (LPS) as demonstrated in the rat (36). Pertaining to the thermogenic function, there is a long controversy as to whether UCPs, in particular UCP1, are present in marsupials and monotremes. In eutherian mammals nonshivering thermogenesis in BAT depends on UCP1-mediated uncoupling of respiration, but it is unclear whether this thermogenic mechanism is also utilized by marsupials and monotremes. Indeed, small body size and the seasonal fluctuations in environmental conditions of marsupial and monotreme habitats, e.g., low temperature and food shortage in the dry season, impose considerable requirements for physiological adaptation. Support for the presence of nonshivering thermogenesis in marsupials was obtained by several physiological studies which demonstrated adrenergic stimulation of resting oxygen consumption in several marsupial species (9, 12, 38, 42–44, 49, 50, 56, 63). Furthermore, GDP binding to interscapular adipose tissue mitochondria of Bennett’s wallaby appeared to support the presence of UCP1 in marsupials (38). However, UCP2 and UCP3 also bind nucleotides, albeit with different affinity and specificity compared with UCP1 (14–16). Other molecular studies addressed the issue by immunologic detection of UCPs (25, 27) and PCR-based strategies (27). Prior to the discovery of the novel UCPs, Hope and coworkers (25) found in the interscapular adipose tissue depot of Sminthopsis crassicaudata a very weak signal corresponding to the molecular weight of UCP1 using an antibody raised against ground squirrel UCP1. At that time cross-reactivity with novel UCPs or other mitochondrial anion carriers could not be excluded. By Northern blot analysis using a rat UCP1 cDNA, no UCP-like transcript could be identified in adipose tissue of macropodids (50). Attempts to gather molecular evidence for the presence of UCP2 and UCP3 orthologs in marsupials have been reported, but no study so far has published sequence data (9, 26, 27).

The objective of our study was to identify UCPs in different marsupial species (yellow-footed antechinus Antechinus flavipes, stripe-faced dunnart S. macroura, common brushtail possum Trichosurus vulpecula) and in the monotreme short-beaked echidna Tachyglossus aculeatus. The small nocturnal marsupial mammals span a body mass range from 15 g to 4.5 kg, whereas the monotreme T. aculeatus may weigh up to 7 kg (57). Two marsupial UCPs were identified and annotated as UCP2 and UCP3 by phylogenetic analysis. We chose the small carnivorous marsupial subspecies A. flavipes flavipes, whose weight ranges from 20 to 50 g in females and up to 80 g in males, to further investigate the regulation of UCP2 and UCP3 gene expression in response to cold exposure and food deprivation in selected tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

Animal experiments.
All experiments had been approved by the University of Southern Queensland Animal Ethics Committee (01REA121), National Parks Queensland and Environment Australia (PWS P2001585). Eighteen yellow-footed antechinus (A. f. flavipes) were captured with Elliott traps from several subtropical habitats in southeast Queensland (Australia) and housed individually for at least 7 days at 24°C (12:12 light/dark, lights on 0700 h) with free access to food and water (cat food pellets soaked in water, and mealworms). To investigate the effect of cold exposure, four individuals of A. flavipes were transferred to a climate chamber at 5°C for 2 days, whereas three controls remained at 24°C. Eight A. flavipes were food-deprived for 48 h, of which three animals were refed 24 h before tissue dissection. A stripe-faced dunnart (S. macroura) was housed in the animal facility of the University of Southern Queensland >6 mo prior to tissue dissection. In addition, road kills of a common brushtail possum (T. vulpecula) and an echidna (T. aculeatus) were collected from highways in the Darling Downs area.

Tissue dissection.
After anesthesia, multiple tissues of A. flavipes (adipose tissue, heart, skeletal muscle, kidneys, liver, lung, stomach, and brain) were immediately sampled and snap frozen in liquid nitrogen. Blood was collected from individuals anesthetized with CO2 by puncture of the vena cava posterior, then centrifuged at 16,000 g for 2 min, and sedimented blood cells were stored at –70°C. We also dissected spleen from T. vulpecula, spleen and interscapular adipose tissue from S. macroura, and adipose tissue, skeletal muscle, liver, kidneys, and spleen from T. aculeatus. All tissue samples were stored at –70°C and shipped in liquid nitrogen to Marburg, Germany. For DNA and RNA extractions all tissue samples were first powdered in liquid nitrogen.

Southern blot analysis.
DNA was isolated from spleen and kidney tissue powder using a standard phenol-chloroform extraction protocol. Tissues were digested with 0.5 mg/ml proteinase K (Sigma) in NTES buffer (50 mM Tris·HCl, 100 mM EDTA pH 8.0, 100 mM NaCl, 1% SDS) at 55°C overnight and centrifuged for 5 min at 2,500 g. The clear supernatant was then incubated for 30 min with an equal volume of neutralized phenol-chloroform-isoamyl alcohol (25:24:1). After centrifugation (15 min, 14,000 g), the DNA (aqueous phase) was precipitated with 2 vol of ethanol and 1/10 vol of sodium acetate (3 M, pH 5.2) for 5 min at room temperature. The DNA was collected by centrifugation for 10 min at 14,000 g, washed in 70% ethanol, and dissolved in appropriate restriction buffers (Amersham). Twenty micrograms of photometrically quantified DNA was digested with AvaII, EcoRI, SmaI, XbaI, SacI, and BamHI (Amersham), subjected to electrophoresis in a 1% TAE-agarose gel, transferred in 20x SSC (1.5 M NaCl, 0.15 M sodium acetate, pH 7.0) to a nylon membrane (Hybond N+, Amersham), overnight and immobilized by UV cross-linking (UV-Stratalinker, Stratagene).

Northern blot analysis.
Total RNA was isolated with TRIzol (GIBCO-BRL) according to the manufacturer’s protocol (8). As an additional step, the RNA pellet was redissolved in a solution containing 6.3 M guanidinium thiocyanate, 40 mM sodium citrate pH 7, 0.8% sarcosyl, 8 mM 2-mercaptoethanol, precipitated with 1 vol isopropanol, washed in 75% ethanol, and finally dissolved in DEPC-treated water. Total RNA was photometrically quantified at 260 nm and stored at –70°C. Twenty micrograms of RNA was electrophoresed in a 1% denaturing agarose gel (5% formaldehyde, 0.02 M MOPS, 5 mM sodium acetate, 1 mM disodium EDTA, pH 8), transferred overnight in 10x SSC to a nylon membrane (Hybond N, Amersham), and UV cross-linked.

Reverse transcriptase-polymerase chain reaction.
RNA was isolated from skeletal muscle of A. flavipes and used for first-strand cDNA synthesis (SuperScript II, GIBCO-BRL) according to the manufacturer’s protocol. Consensus primers were obtained from MWG Biotech, Ebersberg, Germany (sense, 5'-CTG CAG CGC CAG ATG AGC TTC GCC; antisense, 5'-GTT CAT GTA CCG GGT CTT CAC CAC ATC C). 40 cycles of denaturation at 94°C (1 min), annealing at 56°C (1 min), and extension at 72°C (1 min) were performed. A final extension at 72°C was applied for 10 min and followed by rapid cooling to 4°C. The PCR product was gel-purified and ligated into a pGEMT-easy vector (Promega).

cDNA libraries.
Poly(A)+ mRNA was isolated by annealing biotin-labeled oligo(dT) primers coupled to beads (Oligotex mRNA kit Midi, Qiagen, Germany). Double-stranded cDNA was synthesized from 5 µg mRNA and unidirectionally inserted into a bacteriophage vector using the Zap Express XR Library Construction Kit (catalog no. 200451; Stratagene, La Jolla, CA). The libraries were screened for UCP homologs on nylon membranes using probes for UCP1, UCP2, and UCP3. Vectors containing positive cDNA inserts were processed into phagemids according to the manufacturer’s protocol (55). Inserts were sequenced using vector-based primers. The full-length sequence was obtained by primer walking (MWG Biotech) and analyzed using BLAST software (GenBank).

Radioactive hybridization.
cDNAs were random prime-labeled with [{alpha}-32P]dCTP (Rediprime DNA labeling system, Amersham). Nylon membranes were prehybridized at 63°C with BSA solution (0.5M Na2PO4/NaH2PO4, pH 7.0, 1 mM EDTA, pH 8.0, 7% SDS, 1% BSA) for at least 1 h and hybridized overnight at 63°C with the 32P-labeled probe. After hybridization, the blots were washed with 2x SSC/0.1% SDS for 20 min, 1x SSC/0.1% SDS for 10 min, and 0.5x SSC/0.1% SDS for 10 min at room temperature. Signal intensities were then monitored by exposure to a PhosphorScreen (Molecular Dynamics). The stringency of washing was subsequently increased to 0.1x SSC/0.1% SDS for 10 min at 60°C for the A. flavipes UCP2 probe. The hybridized probes were then detected by phosphor imaging (Storm 860, Molecular Dynamics), and signal intensities were quantified using ArrayVision 7.0 (Imaging Research). In the fasting-refeeding experiment UCP2 mRNA in skeletal muscle of A. flavipes was detected by autoradiography at –80°C for 2 days (X-OMAT AR-Film, Kodak) and quantified densitometrically (Scion Image Software 4.0.2).

All blots were hybridized with probes corresponding to the cDNA sequences of rat UCP1 (1,200 bp; GenBank accession number NM_012682.1), mouse UCP2 (1,000 bp; GenBank BC012967.1) and hamster UCP3 (826 bp; GenBank AF271265). The full-length UCP2 insert obtained from the spleen cDNA library of A. flavipes was 32P-labeled and used for Northern blot analysis of gene expression in selected tissues. In addition, RNA extracted from the heart and skeletal muscle of A. flavipes was hybridized with hamster PDK4 cDNA (482 bp; GenBank AF321218).

Ethidium bromide staining of total RNA and hybridization with hamster 18S rRNA and mouse ß-actin served to normalize gel loading. In between subsequent hybridizations, membranes were stripped by shaking for 20 min at 64°C after addition of boiling 0.1x SSC. Data shown in columns represent mean values.

Phylogenetic inference.
A comprehensive search for UCP sequences was performed in public databases using keywords and BLAST alignments (GenBank/EMBL and SwissProt). An alignment of the UCP amino acid sequences was generated using ClustalX 1.81 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX). Program modules of the PHYLIP software package were employed for cladistic analysis. For bootstrapping, the aligned data set was shuffled randomly 1,000 times (SEQBOOT) (19). Distances between pairs of protein sequences were calculated according to Dayhoff’s empirical PAM matrix (13) using the PROTDIST and scaled in expected historical events per site with gaps considered as missing data. A phylogenetic tree was constructed by the neighbor joining method with PROTPARS (51). A majority-rule consensus tree was generated with CONSENSE. The oxaloacetate-malate carrier, considered as an "ancient" branch of the mitochondrial anion carrier family, was used as the outgroup. The choice of other mitochondrial anion carriers, namely "UCP4," "UCP5," ADP-ATP-translocase, or phosphate carrier protein, as the outgroup had no effect on the phylogenetic relationships within the core UCP family.

Statistical analysis.
Statistical analysis was performed using SPSS 11.0 (SPSS Inc.). The effect of food deprivation and refeeding on the expression of UCP2 and UCP3 in different tissues was assessed by the nonparametric Kruskal-Wallis H-Test. The Mann-Whitney U-test was applied for two-sample comparisons. Results were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The presence of UCP-related genes in marsupials and monotremes was first analyzed by Southern blot hybridization of genomic DNA with heterologous rodent cDNA probes for UCP1, UCP2, and UCP3. Mouse UCP2 cDNA clearly hybridized with several genomic restriction fragments of the marsupials A. flavipes and T. vulpecula (Fig. 1). A positive result for UCP2 was also obtained with DNA from S. macroura (data not shown) but not with DNA of the monotreme T. aculeatus (Fig. 1). Initial evidence for UCP2 gene expression in spleen of A. flavipes and S. macroura was obtained by Northern blot analysis using a heterologous mouse UCP2 probe. A single band was detected at a size corresponding to rodent UCP2 mRNA. Despite positive control hybridizations of mouse and hamster DNA with UCP1 or UCP3 probes, no signals were detected on marsupial or monotreme DNA. Similar results were obtained in this analysis when DNA was digested with other restriction enzymes (data not shown).



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  		Fig. 1. Southern blot analysis of genomic DNA from several marsupial species and a monotreme. Twenty micrograms genomic DNA was digested with AvaII and hybridized with either rat uncoupling protein 1 (UCP1), mouse UCP2, or hamster UCP3 cDNA probes. Mouse and hamster DNA served as control. Arrows indicate specific signals. Psu, Phodopus sungorus; Mmu, Mus musculus; Afl, Antechinus flavipes; Tac, Tachyglossus aculeatus; Tvu, Trichosurus vulpecula.

 
As Southern and Northern blot hybridizations with heterologous probes were not successful for UCP3, we tried an alternative approach. We hypothesized that eutherian UCP3 and avian UCPs are orthologs and therefore selected consensus primers from conserved regions for RT-PCR experiments. We amplified a 480-bp cDNA fragment by RT-PCR from skeletal muscle of A. flavipes, which showed high similarity to mouse UCP3. We then generated cDNA libraries from the marsupials S. macroura (spleen) and A. flavipes (spleen and skeletal muscle) to isolate full-length cDNAs of the marsupial UCP2 and UCP3.

Screening of the spleen cDNA libraries with a mouse UCP2 probe identified a 1,576-bp full-length clone from A. flavipes (GenBank AY233003) and a 1,076-bp fragment including a 5' truncated partial coding sequence from S. macroura (GenBank AY232996). The deduced amino acid sequences of these marsupial UCP2 proteins are shown in Fig. 2. The open reading frame (ORF) of the cDNA from A. flavipes encodes a 310-amino acid protein. A GenBank query of both protein sequences revealed an average identity of 88–90% to rodent UCP2.



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  		Fig. 2. Amino acid sequence alignment of UCP2 from Sminthopsis macroura (GenBank accession no. AY232996), A. flavipes (GenBank AY233003), and M. musculus (GenBank AAB17666), UCP3 from A. flavipes (GenBank AY519198) and M. musculus (GenBank PP56501), and UCP1 from M. musculus (GenBank P12242) and Suncus etruscus (30) using ClustalX 1.81 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX) and illustrated with GeneDoc 2.601 (http://www.psc.edu/biomed/genedoc/). Sequences are presented in single letter code. Gaps were introduced by ClustalX to optimize alignment score. Most conserved amino acids are highlighted in black; less conserved amino acids are in brighter background. Putative transmembrane regions are boxed, with arrows indicating amino acids involved in pH sensing of the nucleotide binding (31). H145 and H147, two amino acids involved in the proton transport of UCP1, are boxed and indicated with "H+" (2). Black bars above the alignment indicate the energy transfer protein signature; the black bar below indicates the position of a potential nucleotide binding domain (*) found in UCP1 (6). Sma, S. macroura; Afl, A. flavipes; Mmu, M. musculus; Hsa, Homo sapiens; Set, S. etruscus.

 
Screening the A. flavipes skeletal muscle cDNA library with the 480-bp UCP3 cDNA fragment identified a full-length UCP3 cDNA (GenBank AY519198). The deduced 312-amino acid protein exhibits 80% identity to mouse UCP3. Both marsupial UCPs showed an average identity to rodent UCP1 of 54–55%.

The sequence alignment with other UCPs demonstrates high conservation of the transmembrane domains, the nucleotide binding site (6), and associated pH sensors (E190 and H214) (31) (Fig. 2). The two histidine residues corresponding to H145 and H147 in hamster UCP1, which have been considered to be crucial for proton transport activity of UCP1 (2), are not present in marsupial UCP2 and UCP3.

For classification of marsupial UCP proteins and their relationship to other homologs, we performed phylogenetic analysis and generated a tree (Fig. 3). We retrieved 41 sequences from the core UCP family including plant UCPs from public databases, according to Borecky et al. (3). In our tree, the two marsupial UCP2 and the UCP3 cloned in the present study group together with their respective orthologs and are positioned at the base of the mammalian branch. The identity of marsupial and fish UCP2 is 80%. Our comprehensive search for UCPs further revealed so far unnamed protein products of the amphibians Xenopus laevis (GenBank AAH44682.1) and Silurana tropicalis (GenBank AAH63352), which we identified as UCP2. These amphibian sequences exhibit 81% identity to marsupial UCP2.



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  		Fig. 3. Phylogenetic relationships of the UCP family including UCP2 and UCP3 of A. flavipes and UCP2 of S. macroura. A neighbor joining tree was derived from the amino acid alignment. Bootstrap values from 1,000 replications are given next to the internal branches. *Partial amino acid sequences. **Previously unnamed protein products, which we identified as members of the UCP superfamily. The oxaloacetate-malate carrier served as the outgroup. Afl, A. flavipes; Ath, Arabidopsis thaliana; Bta, Bos Taurus; Cca, Cyprinus carpio; Cfa, Canis familiaris; Dgr, Dicrostonix groenlandicus; Dre, Danio rerio; Ema, Eupetomena macroura; Gga, Gallus gallus; Hmu, Helicodiceros muscivorus; Hsa, H. sapiens; Les, Lycopersicon esculentum; Mac, Macaca mulatto; Mau, Mesocricetus auratus; Mga, Meleagris gallopavo; Min, Mangifera indica; Mmu, M. musculus; Ocu, Oryctolagus cuniculus; Osa, Oryza sativa; Psu, P. sungorus; Rno, Rattus norvegicus; Set, Suncus etruscus; Sfo, Symplocarpus foetidus; Sma, S. macroura; Ssc, Sus scrofa; Str, Silurana tropicalis Stu, Solanum tuberosum; Tae, Triticum aestivum; Xla, Xenopus laevis; Zmy, Zea mays. See the Supplementary Material (available online at the Physiological Genomics web site) for accession numbers of the sequences.

 
Further analysis of the untranslated regions (UTR) of the A. flavipes marsupial UCP2 cDNA identified a second ORF in the 5'-UTR encoding for a putative peptide of 36 amino acids. This ORF contains three start codons (AUG) in frame. By alignment of all available 5'-UTRs from other UCP2 mRNAs, we observed a high degree of conservation of this ORF across vertebrates (Fig. 4). The marsupial UCP2 upstream ORF (uORF) exhibits 79–81% nucleotide identity and 66–69% amino acid identity to the ORF of eutherian mammals.



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  		Fig. 4. Upstream open reading frame (ORF) found in the 5'-UTR of UCP2 mRNA from A. flavipes. The nucleotide sequence was aligned with all available UCP2 full-length mRNAs, and potential start cons (ATG) are boxed (A). The ORF was translated and aligned (B). Conserved nucleotides are highlighted with dark background. Mmu, M. musculus; Hsa, H. sapiens; Rno, R. norvegicus; Afl, A. flavipes; Dre, D. rerio; Cca, C. carpio.

 
To investigate tissue-specific expression of UCP2 and UCP3, we analyzed total RNA from selected tissues by Northern blotting using the homologous A. flavipes cDNA probe. We detected high levels of UCP2 mRNA as a single band at ~1.7 kb in adipose tissue, lung, spleen, and blood cells. UCP2 mRNA was barely detectable in liver, skeletal muscle, heart, and stomach and not detectable in brain (Fig. 5). The highest abundance of UCP2 mRNA was observed in blood cells. Quantitative analysis demonstrated a 3.6-fold higher concentration of UCP2 mRNA in blood cells than in spleen and an even 4.5-fold higher concentration compared with lung and adipose tissue. UCP2 mRNA was also detected in spleen of S. macroura, T. aculeatus (Fig. 5), and T. vulpecula (data not shown), and in the monotreme, weak hybridization signals were observed in adipose tissue and liver. In contrast to this evidence for UCP2, no UCP3 mRNA was detectable with the A. flavipes UCP3 cDNA probe in any tissue of the monotreme (data not shown).



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  		Fig. 5. Northern blot analysis of UCP expression in multiple tissues of the marsupials A. flavipes (UCP2 and UCP3) and S. macroura (UCP2) and the monotreme, the short-beaked echidna T. aculeatus (UCP2). Total RNA (20 µg) isolated from selected tissues was hybridized with the A. flavipes full-length UCP2 cDNA and A. flavipes UCP3 cDNA fragment. Total RNA from hamster brown adipose tissue served as a control.

 
The expression of UCP3 mRNA in A. flavipes is restricted to heart and skeletal muscle (Fig. 5). As in rodents, the mRNA levels in heart were minor compared with skeletal muscle. In contrast to the mRNA isoforms known from rodents, only one splicing variant is visible on the blot, and the size corresponds to the size of UCP2 mRNA.

It has been proposed that novel UCPs could have a thermogenic function in marsupials, which most likely lack UCP1 (28, 50). Otherwise, UCP2 and UCP3 have been implicated in lipid mobilization and utilization in response to fasting. Therefore, we investigated the physiological regulation of UCP gene expression in selected tissues of A. flavipes exposed to cold and in response to fasting/refeeding. Cold exposure had no significant effect on UCP2 mRNA level in adipose tissue, and UCP2 as well as UCP3 mRNA were barely detectable in skeletal muscle of warm and cold exposed A. flavipes (data not shown).

In response to fasting, UCP3 mRNA level was 2.5-fold and 6-fold increased in skeletal muscle and heart, respectively, which was reversed upon refeeding for 24 h (Fig. 6, A and B). PDK4 mRNA levels in skeletal muscle and heart, taken as an index for the switch from carbohydrate to fatty acid metabolism in the fasted state, showed a threefold and sevenfold increase in skeletal muscle and heart, respectively (Fig. 6, A and B). UCP2 expression in response to fasting was decreased in lung and increased in adipose tissue but unaffected in spleen and skeletal muscle (Fig. 6C).



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  		Fig. 6. Northern blot analysis in response to fasting in A. flavipes of UCP2, UCP3, and PDK4 mRNA in skeletal muscle (A), UCP3 and PDK4 mRNA in heart (B), and UCP2 in lung, adipose tissue, and spleen (C). Twenty micrograms total RNA from selected tissues of ad libitum-fed, food-deprived, and refed A. flavipes was hybridized with the A. flavipes full-length UCP2 cDNA and a 480-bp UCP3 cDNA fragment. Right: diagrams show individual UCP2 intensities (dots) and mean values of the ad libitum-fed group (open bar), 48-h fasted group (dark gray bar), and 24-h refed group (light gray bar). All intensities were normalized with either 18S rRNA or ß-actin intensities. aP < 0.05 for fasted vs. fed ad libitum; bP < 0.05 for fasted vs. refed (Mann-Whitney U-Test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Previous studies provided suggestive evidence for the presence of uncoupling proteins in marsupial mammals, but unambiguous molecular proof for their existence is lacking in metatheria as well as in prototheria. We here report on the sequence and tissue-specific expression pattern of marsupial UCP2 and UCP3.

First results suggesting the presence of UCP2 in metatheria and prototheria were obtained by comparative Southern and Northern blot analysis, whereas this approach was not successful for UCP1 and UCP3. We suggested a close relationship between avian UCPs and mammalian UCP3 based on the similar tissue-specific expression and physiological regulation (10, 17) and succeeded to amplify a 480-bp UCP3 cDNA fragment from skeletal muscle of A. flavipes using consensus primers.

Screening of cDNA libraries led to the identification of marsupial UCP2 (spleen, A. flavipes and S. macroura) and UCP3 (skeletal muscle, A. flavipes) (Fig. 2). In both proteins, transmembrane domains found in other members of the UCP family and several functional residues originally identified in UCP1 involved in pH sensing and nucleotide binding are conserved. This high degree of identity of marsupial and rodent UCP2 and UCP3 suggests a conserved function of the proteins in both mammalian subclasses.

We generated a phylogenetic tree with 44 sequences of UCP family members including the new marsupial UCP sequences and two previously nonannotated UCPs. UCP2 and UCP3 of S. macroura and A. flavipes are positioned at the base of their mammalian orthologs (Fig. 3). 1 Addition of the A. flavipes UCP3 sequence stabilized the node leading to the distinction between UCP2 and UCP3 sequences from 59% to 68% in our analysis. Beyond the comparison of marsupial UCPs with other mammalian orthologs, the phylogenetic analysis illustrates the broader relationships of UCP family members and enables the annotation of previously unnamed protein products. The present analysis provides the first demonstration of UCP2 in the animal class Amphibia.

The presence of UCP1 in marsupials remains unknown. Based on the phylogenetic tree, hypothesis can be generated on the presence of other UCPs in proto- and metatheria. Pertaining to UCP1, the presented tree questions the long-standing point of view that this protein is only found in placental mammals. Taking in account, the position of the node leading to the UCP1 cluster as well as the presence of UCP2 in fish and amphibians suggests that UCP1 (and UCP3) already exist(s) in lower vertebrates. Despite a high phylogenetic distance between amphibians, fish, and rodents, the UCP2 orthologs are very similar (~80%). In contrast, the comparison of UCP1 orthologs from phylogenetic distant species within eutherian mammals, as exemplified by shrew vs. mouse UCP1, reveals only 75% global identity based on 245 amino acids known from shrew. This indicates a rapid evolution of this protein within the eutherian infraclass. This may be one major complication in the attempt to determine the existence of an UCP1 ortholog in nonplacental mammals.

We searched for conserved sequence motifs in the UTR of the A. flavipes UCP2 transcript, as others have demonstrated the presence of regulatory elements involved in translational regulation of the transcript (46). This led to the identification of a uORF in the 5'-UTR (Fig. 4). Surprisingly, the marsupial uORF is almost identical to an upstream ORF identified by Pecqueur and coworkers (47) in the transcripts of the mouse and human UCP2 genes. Functional analysis in cell systems revealed an inhibitory effect of the uORF on translation efficiency that depends on the utilized cell line (46, 47). The high level of conservation of this upstream ORF in metatheria emphasizes the functional significance for the regulation of UCP2 synthesis. Furthermore, we identified this uORF in the UCP2 full-length sequences of rat, zebrafish, and carp. So far there is no evidence for translation of this uORF. In all mammals, three AUG triplets are positioned in identical position in the uORF, whereas in fish, only two of them can be found (Fig. 4). The translation of eukaryotic mRNA is dependent on structures of the leader and the Kozak sequence (33, 45). Elements of the 5'-UTR control protein synthesis and especially upstream AUGs correlate negatively with translation efficiency (22, 32, 39, 48). Binding of ribosomes to uAUGs instead of the actual translational start codon would result in a lower translation rate. This control element for translational efficiency seems to be conserved during the evolution of vertebrates.

Following the unequivocal demonstration of UCP2 and UCP3 in marsupials, we subsequently investigated in A. flavipes the tissue-specific expression pattern and physiological regulation of gene expression in response to cold exposure and fasting by selecting tissues in which these physiological stimuli have been reported to alter the expression of UCP2 and UCP3 in mice and rats. Tissue specificity of UCP2 and UCP3 gene expression in marsupials is very similar to placental mammals (40). A. flavipes UCP3 mRNA is restricted to heart and skeletal muscle with higher levels in the latter tissue. In contrast, a ubiquitous tissue distribution of UCP2 mRNA was found in A. flavipes as described previously in rodents (Fig. 5), with strongest expression levels observed in tissue sites rich with macrophages, like lung and spleen (20, 59). We detected the highest abundance of UCP2 mRNA in blood cells. This is consistent with findings in rodents and humans where high levels of UCP2 expression have been observed in monocytes and macrophages (34). However, we analyzed RNA from the total blood cell population in which white blood cells make up only 0.1% of the total cell number, and it is unlikely that erythrocytes and blood platelets, which lack mitochondria, express significant amounts of UCP2. Thus, if UCP2 expression is restricted to white blood cells, then the cell-specific mRNA abundance must be extremely high (Fig. 5). In blood and macrophage-rich tissues the level of UCP2 expression appears to regulate the generation of ROS, indicating a function in the modulation of the mammalian immune system. UCP2 knockout mice gain resistance against Toxoplasma gondii infection (1), and downregulation of UCP2 in macrophages promotes the generation of bactericidal substances (29).

UCP2 and UCP3 have been proposed to be either related to thermogenesis, lipid oxidation, or ROS defense (15, 21). However, in accordance with rodent studies, cold exposure for 2 days affected neither UCP2 (adipose tissue, skeletal muscle) nor UCP3 (skeletal muscle) mRNA levels in A. flavipes. In only one study of rat soleus muscle was an increase of UCP2 mRNA level in response to cold reported (4), but the functional significance has not been resolved.

We, as well as others, have failed to demonstrate the presence of UCP1 in marsupials, but it has been speculated that marsupial UCP2 and UCP3 could be involved in nonshivering thermogenesis (28). However, based on the lack of cold-induced expression in adipose tissue and skeletal muscle, such a thermogenic function of UCP2 and UCP3 appears unlikely. In the mouse only UCP1 ablation results in a defect of adaptive thermogenesis in the cold (23). Furthermore, the two-histidine motif conserved in UCP1 (H145/H147 in hamster) and involved in the unique proton transport capacity is not present in rodent UCP2 and UCP3 (2). Therefore, it was suggested that only UCP1 is able to uncouple the respiratory chain. However, the functional significance of the two-histidine motif must be questioned, as H145 is not conserved in shrew UCP1 (Fig. 2). Moreover, Echtay and coworkers (14–16) found that superoxides and coenzyme Q not only stimulate the uncoupling activity of UCP1, but also activate UCP2 and UCP3. Even if marsupial UCP2 exhibits uncoupling activity, it is not induced in response to cold.

In eutherian mammals UCP2 and UCP3 expression is increased in response to fasting in skeletal muscle (4, 7, 52, 53, 60). In fasted A. flavipes UCP3 mRNA level is increased in skeletal muscle and in heart and diminished by refeeding (Fig. 6). Interestingly, physiological regulation of UCP2 expression in skeletal muscle of A. flavipes in fasting/refeeding was absent. In this fasting/refeeding experiment PDK4 expression, taken as a marker for a metabolic switch toward lipid oxidation (61, 62), closely resembled the changes in UCP3 mRNA levels. This suggests that UCP3 is recruited when muscle metabolism preferentially oxidizes lipids. Recent reports substantiate the hypothesis that UCP3 may function as a fatty acid anion exporter, thereby preventing the accumulation of nonesterified fatty acids in the mitochondrial matrix (24, 54). In conclusion, the regulation of skeletal muscle UCPs (avian UCP, marsupial UCP3, and placental UCP3) in response to fasting and refeeding across a broad range of endotherms suggests an identical physiological function.

UCP2 mRNA level was significantly decreased in lung and increased in adipose tissue but not altered in spleen and skeletal muscle (Fig. 6). Regulation of UCP2 expression in lung and adipose tissue has not been reported for rodents (46), and the functional significance of this interspecific difference is unclear. However, UCP2 mRNA expression does not reflect UCP2 protein levels, which have been shown to increase in the absence of mRNA regulation (49). Furthermore, the high UCP2 mRNA levels found in blood cells may have to be considered in this respect. Results obtained in UCP2 gene expression studies using total tissue RNA may be affected by tissue blood volume. Alteration of tissue blood volume in response to physiological challenges like fasting or cold exposure may result in apparent changes in UCP2 gene expression. In situ hybridization studies, as already published for UCP2 in the brain, could rule out this possible source of error in future experiments.

In summary, we unequivocally identified UCP2 and UCP3 in marsupials and provided first evidence for the expression of UCP2 in a monotreme. Sequence elements of the marsupial UCPs are strongly conserved during evolution of the 130 million year old marsupial lineage, suggesting an identical protein function that is further corroborated by the similar tissue expression pattern and physiological regulation of gene expression in marsupials and placental mammals. We found no indication for the involvement of marsupial UCPs in adaptive nonshivering thermogenesis. Further comparative analysis of UCP2 and UCP3 from evolutionary distinct species will assist in testing different hypotheses pertaining to the functional annotation of these mitochondrial anion carrier proteins.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by Deutsche Forschungsgemeinschaft DFG KL973/7 (to M. Klingenspor) and by a grant to Kerry Withers from the Department of Biological and Physical Sciences, University of Southern Queensland.


   ACKNOWLEDGMENTS
 
We are grateful to the University of Queensland, Dr. Geoff Lundie-Jenkins from Queensland Parks, Environment Australia, and the staff of the Department of Natural Resources, Inglewood, for use of their facilities. We also want to thank Queensland Museum for providing us with tissues from a variety of Australian marsupials and several Queensland farmers for permission to trap on their properties.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: M. Jastroch, Philipps-Univ. Marburg, Dept. of Biology, Animal Physiology, Karl-von-Frisch-Str. 8, 35043 Marburg, Germany (E-mail: jastroch{at}staff.uni-marburg.de).

10.1152/physiolgenomics.00165.2003

1 The Supplementary Material for this article is available online at http://physiolgenomics.physiology.org/cgi/content/full/00165.2003/DC1. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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