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J. Biol. Chem., Vol. 275, Issue 46, 36394-36399, November 17, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/46/36394    most recent M005713200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Esterbauer, H. Articles by Patsch, W. Search for Related Content PubMed PubMed Citation Articles by Esterbauer, H. Articles by Patsch, W. Social Bookmarking                      What's this?

The Uncoupling Protein-3 Gene Is Transcribed from Tissue-specific Promoters in Humans but Not in Rodents^*

Harald Esterbauer, Hannes Oberkofler, Franz Krempler^§, A. Donny Strosberg^¶, and Wolfgang Patsch

From the  Department of Laboratory Medicine, Landeskliniken Salzburg, A-5020 Austria, the ^§ Department of Internal Medicine, Krankenhaus Hallein, A-5400 Austria, and the ^¶ Institute Cochin de Genetique Moleculaire, Paris, 75014 France

Received for publication, June 29, 2000, and in revised form, August 23, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Uncoupling protein-3 (UCP3), a mitochondrial membrane transporter, is a candidate effector of thermogenesis. Even though mice with targeted disruption of the UCP3 gene are not obese, indirect evidence suggests that this protein contributes to the control of energy expenditure in humans. We therefore characterized the human UCP3 gene and compared it with its rodent homologues with respect to tissue-specific expression and regulatory regions. Like rodent UCP3, human UCP3 was expressed in skeletal muscle and brown adipose tissue (BAT). The short mRNA isoform, UCP3_S, which is absent in rodents, was relatively more abundant in human skeletal muscle in comparison to human BAT. Two tissue-specific transcription start sites for each skeletal muscle and BAT were delineated for human UCP3. Tissue-specific transcript initiation was maintained in both tissues and cultured cells over a wide range of expression levels. In contrast, rodent transcripts were initiated at the same site in BAT and muscle tissue. Comparison of human and rodent promoters indicated a rapid phylogenetic evolution suggesting functional diversification. The transcription from tissue-specific promoters in humans is a novel finding that may provide the basis for therapeutic interventions aimed at regulating energy expenditure in a tissue-specific fashion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Uncoupling protein-3 (UCP3),¹ a member of the mitochondrial carrier superfamily, displays 56 and 73% homology to UCP1 and UCP2, respectively (1). UCP1 is exclusively expressed in brown adipose tissue (BAT), which is specialized in adaptive thermogenesis. UCP2 is expressed in several tissues including BAT (2, 3), and in rodents, high expression levels of UCP3 mRNA are observed in both skeletal muscle and BAT (4-6). UCP1 plays a central role in BAT function by uncoupling proton entry in the mitochondrial matrix from ATP synthesis, thereby generating heat (7). BAT may contribute to the regulation of total body fat stores in rodents, since mice made deficient in BAT through a transgenic approach using UCP promoter-driven diphtheria toxin A developed marked obesity (8). However, these BAT-deficient mice also displayed extreme leptin resistance (9). Moreover, transgenic mice expressing UCP1 from the fat-specific aP2 promoter exhibited an even greater loss of BAT but were resistant to diet-induced obesity, presumably due to ectopic expression of UCP1 in white fat (10). Thus, the relationship of BAT deficiency or UCP1 deficiency with obesity remains to be established.

Skeletal muscle accounts for a large portion of catecholamine- and diet-induced thermogenesis in both humans and rats (11, 12). Even though the functions of UCP2 and UCP3 have not been delineated, transfection studies in yeast and experiments with reconstituted proteoliposomes clearly demonstrated that both proteins have proton transport activities and may therefore be candidate effectors of thermogenesis (2-4, 13-15). The UCP2 and UCP3 genes are located in adjacent regions of human chromosome 11q13 (16) and mouse chromosome 7 (17). Associations of markers in the vicinity of the UCP2/3 gene locus with resting metabolic rate and regional fat mass have been demonstrated in the Quebec Family Study (18). This locus has also been linked with quantitative trait loci for obesity in rodents (19, 20) and the insulin-dependent diabetes locus-4 in humans (21, 22). Muscle UCP3 expression may be a determinant of energy expenditure in non-diabetic Pima Indians (23). Moreover, UCP3 mRNA levels were reduced in skeletal muscle from type 2 diabetic subjects and were correlated with insulin-mediated glucose utilization in diabetics in some (24), but not in all, studies (25, 26). Owing to alternative polyadenylation/splicing, human UCP3 mRNA occurs in two isoforms termed UCP3_L and UCP3_S (16). African Americans and tribesmen from Sierra Leone with a deficiency of UCP3_L due to an exon 6-splice donor stop polymorphism showed an elevated respiratory quotient and reduced fat oxidation in comparison to wild-type subjects (27). These results that supported a role of UCP3_L in fuel metabolism are at variance with findings of another study (28) that found no abnormalities in the respiratory quotient of affected individuals. Finally, two independent studies reported down-regulation of UCP3 mRNA expression in muscle of weight-reduced subjects (29, 30), an observation that could help to explain the reduced energy expenditure that has been reported after weight reduction (31).

Recent studies in mice with targeted disruption of the UCP3 gene suggested, however, that UCP3 was not required for body weight regulation under the conditions studied (32, 33). Whereas skeletal muscle mitochondria of knock-out mice displayed a greater degree of coupling, their only phenotypic abnormality was an increased production of reactive oxygen species. These studies cast doubt on UCP3 null mutations as a cause of monogenic obesity but do not exclude a role of UCP3 in the pathogenesis of polygenic energy and/or metabolic disorders in humans, since UCP3 and its functional demands may differ between mice and humans. To gain insight into possible differences between rodent and human UCP3 regulation, we characterized the human UCP3 gene in detail and compared the regulatory regions of the rodent and human UCP3 genes. We report here distinct differences in tissue-specific transcript initiation between humans and rodents, and we provide evidence for a rapid phylogenetic divergence of the UCP3 regulatory regions between humans and rodents.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Skeletal Muscle and Adipose Tissue Samples-- Human tissue samples were obtained during elective surgical procedures. Samples included skeletal muscle from the rectus abdominis and intraperitoneal adipose tissue of 12 subjects. In addition, a perirenal fat biopsy was obtained from a patient with pheochromocytoma. Tissue biopsies were taken at the beginning of the surgical procedure, snap-frozen in liquid nitrogen, and stored at 70 °C until needed. All study subjects provided informed consent, and the study was approved by the institutional review board. Harlan Sprague-Dawley rats and C56BL/6J mice were killed by cervical dislocation. Muscle tissue and BAT samples were collected from the gastrocnemius/soleus and interscapular deposits and immediately frozen in liquid nitrogen.

Isolation of DNA and Total RNA-- Genomic DNA was isolated from human, mouse, and rat leukocytes using the QIAamp Blood Kit (Qiagen, Hilden, Germany). Total RNA was isolated from the respective human and rodent tissues and cultured cells according to the method of Chomczynski and Sacchi (34). All RNA samples were digested with DNase I to eliminate any contaminating DNA. The integrity of RNA samples was ascertained by their electrophoretic pattern in formaldehyde gels. RNA concentrations were determined by absorbance measurements at 260 nm.

5'-Rapid Amplification of cDNA Ends (5'-RACE)-- Transcription initiation sites of human, rat, and mouse UCP3 were determined using the Marathon-RACE kit (CLONTECH, Palo Alto, CA). Adapter-ligated cDNAs were prepared from RNA isolated from human skeletal muscle, intraperitoneal and perirenal adipose tissue, as well as from rat and mouse skeletal muscle and BAT. UCP3 species-specific reverse primers H509R and MR224R (Table I) were used for synthesis of human (H) and mouse/rat (MR) cDNA, respectively. Transcription initiation sites were determined by nested PCR amplification of the respective cDNAs with the adapter primers provided by the manufacturer and two nested UCP3-specific reverse primers, H263R/nested H182R and MR212R/nested MR188R. All PCRs were performed with the Expand High Fidelity System (Roche Molecular Biochemicals). PCR products were cloned into pGEM-T (Promega, Madison, WI), and multiple clones were sequenced from both sides by dye terminator cycle sequencing using an ABI PRISM^TM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Ribonuclease (RNase) Protection Assays-- The UCP3 cDNA probe spanning exons 6 and 7 for the simultaneous quantification of human UCP3_L and UCP3_S transcripts was prepared as described (29). A mouse and rat UCP3 probe that was homologous to the respective human region was constructed using MR808F and MR1167R as primers (Table I). To map transcription start sites in mouse, rat, and human tissues, composite DNA/cDNA probes consisting of UCP3 promoter-exon 1-exon 2 sequences were constructed by an overlap extension approach. A UCP3 promoter-exon 1 fragment was generated by PCR using primer pairs H6527F/H6974R and MR2471F/MR2666R for human and mouse/rat genomic DNA, respectively. A UCP3 exon 1-exon 2 cDNA fragment was amplified by PCR from the respective skeletal muscle RNA using primer pairs H6927F/H263R and MR2620F/MR188R. PCR fragments were gel-purified and mixed to allow annealing of the overlapping exon 1 regions. Subsequent PCR amplification was performed using the respective outer forward and reverse UCP3 primer pairs H6527F/H263R and MR2471F/MR188R. The resulting species-specific composite DNA/cDNA-UCP3 fragments were cloned into pGEM-T (Promega) and verified by sequencing. -³²P-Labeled antisense RNA probes were synthesized from linearized plasmids using the Riboprobe Combination System-SP6/T7 (Promega) and [-³²P]dUTP (800 Ci/mmol; Amersham Pharmacia Biotech). After overnight hybridization of excess -³²P-labeled antisense cRNA with tissue RNA, unprotected RNA was digested with RNase A/T1, and RNA/RNA hybrids were precipitated. Protected fragments were separated by electrophoresis in 4% polyacrylamide-urea gels and visualized by autoradiography. Quantification was by scanning of autoradiographs followed by densitometric analysis (Quantity One Imaging Software, Bio-Rad).

Sequencing and Sequence Analysis of the Rat UCP3 Promoter Region and the Human UCP3 Gene-- For isolation and sequencing of the rat UCP3 promoter region and the human UCP3 gene, the respective Genome Walker kits (CLONTECH) were used as detailed recently (35). Supplied adapter-ligated genomic restriction libraries prepared from rat and human genomic DNA were subjected to PCR using the Expand High Fidelity PCR System protocol (Roche Molecular Biochemicals). Primers specific for rat and human UCP3 were deduced from GenBank^TM accession numbers AF035943 and U84763, respectively. Primer sequences and detailed PCR conditions are available from the authors upon request. PCR products were sequenced from both sides by primer walking. Results from genome walking were verified by genomic PCR. Sequences for the rat UCP3 promoter region and the human UCP3 gene locus were deposited in GenBank^TM.

ClustalW version 1.8 (36) was used for multiple alignment of human, mouse, and rat UCP3 promoter and exon 1 regions. Phylograms were calculated by the Neighbor-joining method (37) with corrections made for multiple substitutions (38) and gaps being included or excluded from the analysis. TreeView (39) was used to display unrooted phylograms. UCP3 promoter-exon 1 similarity plots were produced using the GCG sequence analysis package (Wisconsin Package version 8.0, Genetics Computer Group, Madison, WI). In addition, regions conserved between human, rat, and mouse loci were identified by BLAST searches (40), and interspersed genomic repeats were identified with CENSOR (41). Putative transcription factor binding sites were predicted using the MatInspector program (42) based on the Transfac Data base (43).

Cell Culture Studies-- All drugs and media were obtained from Sigma with the exception of Troglitazone (Sankyo, Tokyo, Japan), the 3-agonist L-755.507 (Merck), and the skeletal muscle cell growth medium (SkGM Bullet Kit; Clonetics, BioWhittaker, Walkersville, MD).

PAZ-6 cells were expanded in DMEM/F-12 medium supplemented with 10% fetal calf serum and 15 mM HEPES. For differentiation, confluent PAZ-6 preadipocytes were cultivated for 8 days in DMEM/F-12 supplemented with 15 mM HEPES, 5% fetal calf serum, 0.1 µM dexamethasone, 850 nM insulin, 1 nM triiodothyronine, and 1 µM troglitazone. During the first 4 days, 0.25 mM 3-isobutyl-1-methylxanthine was included in the differentiation medium (44). Stimulation studies were performed on day 8 after the induction of differentiation. Cells were washed twice with HBSS and incubated for 6 h in DMEM/F-12 supplemented with either 1 µM L-755.507 (Merck) (45) or 1 nM triiodothyronine (T₃).

Human primary skeletal muscle cells were isolated, cultured, and differentiated according to Blau and Webster (46) and Ham et al. (47) using the modifications of Pavlath (48). Satellite cells from biopsies of human rectus abdominis muscle were released from myotubes by trypsinization. Contaminating fibroblasts were removed by selective adhesion of myoblasts to CD56 (Leu19; Becton Dickinson, San Jose, CA), which was immobilized onto magnetic beads (CELLection Pan Mouse IgG Kit; Dynal A.S., Oslo, Norway). The purified myoblast population was plated on collagen-coated dishes and expanded in skeletal muscle cell growth medium (SkGM Bullet Kit; Clonetics). Differentiation of myoblasts was induced at 80% confluence by changing the growth medium to DMEM containing 2% dialyzed fetal bovine serum supplemented with 10 µg/ml insulin. Fusion started on the 1st day and was nearly complete at day 4. Four-day-fused myotubes were used for stimulation experiments with 1 µM isoproterenol or 1 nM T₃ for 6 h as suggested by Henry et al. (49).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Relative UCP3_L and UCP3_S Levels Differ in Human Skeletal Muscle and Adipose Tissue-- To identify UCP3_L and UCP3_S transcripts in human tissues containing or being representative of BAT, RNase protection assays were performed with RNA extracted from intraperitoneal white adipose tissue, perirenal adipose tissue of a patient with pheochromocytoma, and differentiated PAZ-6 cells. The antisense RNA probe used spanned the exon 6-exon 7 splice site and protected two fragments in all human adipose and skeletal muscle tissues studied. The fragments consisted of 354 nt, representing UCP3_L, and 174 nt, representing UCP3_S (Fig. 1). In rat and mouse skeletal muscle and BAT, no UCP3 transcripts homologous to human UCP3_S were found. This result was verified by sequencing of mouse and rat intron 6 lacking a poly(A) signal (not shown) and is consistent with a previous report (17). In humans, UCP3_L/UCP3_S transcript abundance ratios differed between skeletal muscle and adipose tissue obtained from the same individuals in that molar abundance ratios of UCP3_L and UCP3_S transcripts averaged ~1.0 (range 0.9-1.2) in samples representing adipose tissue deposits, whereas UCP3_S was twice as abundant as UCP3_L in the skeletal muscle samples as has been reported previously (29, 50). Recent in vivo studies in humans demonstrated distinct responses of UCP3_L and UCP3_S mRNA levels to triglyceride and glycerol infusions (50), and studies in type 2 diabetics showed that UCP3_L more closely paralleled changes in free fatty acid levels than UCP3_S (25). Thus, the difference in relative abundance levels of UCP3 mRNA isoforms between adipose and muscle tissues of our study subjects may relate to tissue-specific differences in intracellular fatty acid generation. The increase in UCP3 transcript abundance in going from WAT to perirenal fat and PAZ-6 cells is consistent with selective expression of UCP3 mRNA in brown adipocytes. UCP3 mRNA abundance was about 200-400 times lower in intraperitoneal adipose tissue than in skeletal muscle samples. Based on our previous estimate of one brown adipocyte per 200 white adipocytes in human intraperitoneal adipose tissue (51), the order of magnitude of UCP3 transcripts should be comparable between human muscle and brown adipocytes.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Evidence for UCP3 expression in human brown adipocytes and for tissue-specific regulation of UCP3L and UCP3S isoform abundance. Autoradiograph of gel-separated protected fragments in total RNA isolated from intraperitoneal adipose tissue (40 µg), perirenal adipose tissue (10 µg), PAZ-6 cells (20 µg), and skeletal muscle (5 µg) are shown in lanes 1-4. Protected fragments in total RNA extracted from mouse skeletal muscle (5 µg) and BAT (5 µg) are shown in lanes 5 and 6, respectively. Protected fragments representing UCP3L and UCP3S as well as their nucleotide content are indicated on the right.

5'-RACE and RNase Protection Assays Unmask Different Promoter Usage/Transcription Start Sites in Human Skeletal Muscle and Tissues Representing BAT-- 5'-RACE studies revealed two tissue-specific transcription start sites for both skeletal muscle and BAT, represented by intraperitoneal and perirenal adipose tissues and differentiated PAZ-6 cells (Fig. 2). One major initiation site (SM-1), 108 nt upstream of intron 1, and one minor initiation site (SM-2), 151 nt upstream of the major site, were delineated in skeletal muscle. Sequence alignment showed degenerate putative TATA signals, CAP sites, and initiator regions within the expected distance of each of the two start sites. In RNA isolated from perirenal adipose tissues and PAZ-6 cells, the majority of clones obtained by 5'-RACE extended at least 505 nt upstream of the intron 1 border (AT-1) with no clear-cut start site. In addition, several clones were obtained that contained two additional non-coding exons verified by sequencing of PCR products amplified from adipose tissue cDNA. This second adipose tissue transcript initiation site (AT-2) was ~4 kilobase pairs upstream of AT-1. Computational analysis revealed several potential cAMP response elements, binding sites for CRE-binding proteins surrounding AT-2 (42, 43). In contrast to human tissues, rodent tissues including skeletal muscle and interscapular BAT from mice and rats displayed one identical transcription start site (SM/AT) in 5'-RACE experiments. Rodent transcripts initiated ~130 nt upstream of the intron 1 border from a TATA-less promoter.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   5'-RACE reveals distinct UCP3 transcript initiation sites in human skeletal muscle and adipose tissue. Major and minor skeletal muscle and adipose tissue-specific start sites (arrows) are indicated by SM-1 and SM-2 and AT-1 and AT-2. Numbers above arrows show the number of nucleotides relative to the exon 1-intron 1 border. The locations of SM-1, SM-2, AT-1, and AT-2 correspond to nucleotides 6924, 6773, <6527, and ~2558, respectively, in GenBankTM accession number AF050113. Black boxes represent exon 2 containing the translation initiation codon (ATG), and gray boxes represent non-coding exons 1, A and B, as determined by 5'-RACE. Methodological uncertainties in mapping AT-1 are indicated by the hatched portion of the respective exon 1.

5'-RACE results were confirmed by RNase protection assays using as probe a 710-nt fragment that contained 205 nt of exon 2 and extended to 505 relative to the exon 1-intron 1 border. As predicted by 5'-RACE experiments, the pattern of protected fragments differed between human tissues (Fig. 3). In skeletal muscle, two protected fragments were observed. The much more intense and therefore main skeletal muscle transcription initiation site mapped to SM-1. A second, less abundant fragment extended ~150 nt upstream of the main start site and therefore mapped to SM-2. Importantly, neither fragment was observed in adipose tissue. In some experiments, a fragment corresponding to the full-length probe was noted, but the intensity of this band was less than 0.1% of the main fragment, and no evidence for transcript initiation upstream of SM-2 was found in RACE studies. In RNA from adipose tissues, two fragments distinct of those observed in skeletal muscle were protected. The more abundant fragment corresponded to full-length probe. Hence, probes located further upstream were used for mapping of the initiation site. However, due to the presence of a combined L1MB7/MIR1/ALU element in this region (Fig. 4A), precise mapping was impossible. The second, minor start site in adipose tissues (AT-2) corresponded in length to the exon-intron border of an alternatively spliced transcript that was, according to the 5'-RACE results, initiated far upstream of AT-1. These experiments conclusively established tissue-specific UCP3 transcript initiation in humans.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   UCP3 transcript initiation mapping by RNase protection assay in human (A) and mouse (B) skeletal muscle and tissues representative of BAT. A, lanes 1-4, autoradiograph of gel-separated protected fragments in total RNA isolated from human intraperitoneal (40 µg) and perirenal adipose tissue (10 µg), PAZ-6 cells (20 µg), and PAZ-6 cells (20 µg) incubated with the 3-agonist L-755.507 for 6 h. Lanes 5 and 6, protected fragments in total RNA (5 µg) isolated from skeletal muscle of a human subject before and after weight loss. Protected fragments mapping to adipose tissue-specific and skeletal muscle-specific initiation sites are indicated by AT-1, AT-2, SM-1, and SM-2, respectively. B, lanes 1 and 2, autoradiograph of gel-separated protected fragments in total RNA isolated from mouse skeletal muscle and BAT (5 µg each). Protected fragments are indicated by SM/AT. The structural relations between the human or mouse probes and tissue-specific fragments is shown on the right. The human probe spanned 505 nucleotides of promoter-exon 1 (gray box) and 205 nucleotides of exon 2 (black box); the mouse probe spanned 252 nucleotides of promoter-exon 1 (gray box) and 112 nucleotides of exon 2 (black box). The nucleotide distance from individual start sites to the exon 1-intron 1 border is indicated by numbers above black lines, representing the protected fragments.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Large scale alignment (A) and phylogenetic tree (B) of human, rat, and mouse UCP3 5'-regulatory regions. A, large scale alignment. Numbers on the horizontal axis (position) represent distances in nucleotides from the beginning of the aligned file using the exon 1-intron 1 boundary as anchor point for human, rat, and mouse UCP3 sequences. Numbers on the vertical axis represent the proportion of identical nucleotides within a 100-nt window moved across the entire alignment. The dashed horizontal line indicates the average identity (~53%) across the entire alignment of all three species. Average homology of rat and mouse sequences (~83%) is represented by the black line. Black triangles mark human skeletal muscle (SM-1 and SM-2) and adipose tissue (AT-1) transcript initiation sites. The respective mouse/rat start site (SM/AT) is shown as open triangle. The minor adipose tissue start site AT-2 is located upstream of the aligned sequences. Shaded boxes represent the ancient mammalian repeats Alu-Sx and Alu-Jo (Alu family), L1MB7 (LINE family), and MIR1 (MIR family). Black bars indicate the location of human EST clones highly homologous to mouse and rat UCP3 promoter regions. Numbers below bars represent the GenBankTM accession numbers of the respective human EST clones. B, rootless phylogenetic tree based on large scale sequence alignment results. The bar indicates the nucleotide substitution rate per site since the divergence from a putative common UCP3 ancestor sequence located at the branching point.

For mapping of the mouse transcription start site, a probe that contained 112 nt of exon 2 and extended 252 nt upstream of the intron 1 border was used. Mouse BAT and skeletal muscle total RNA showed identical protected fragments confirming the 5'-RACE results. Identical UCP3 transcription start sites were also observed in skeletal muscle tissue and BAT of rats using a rat-specific probe (data not shown).

To determine whether tissue-specific transcript initiation is maintained over a wide range of expression levels, human muscle tissues from obese and weight-reduced obese subjects displaying up to 10-fold differences in UCP3 mRNA levels were used (29). In all samples studied, >98% of the transcripts initiated at SM-1 with the remaining transcripts initiating at SM-2. The effect of known UCP3 stimulants on transcript initiation was studied in primary fused myotubes. Since the UCP3 mRNA level in cultured cells was much lower than in muscle tissue, a semiquantitative reverse transcription-PCR was used to measure mRNA levels and to identify tissue-specific transcripts. Incubation of cells with 1 µM isoproterenol and 1 nM T₃ increased UCP3 mRNA abundance to 160 and 210% of basal levels, respectively (n = 3, p < 0.05). Transcript initiation under basal and T₃- or isoproterenol-stimulated conditions occurred only at SM-1 and SM-2 as judged by the generation of PCR products with SM-1- and SM-2-specific primers and the lack of amplification products with AT-1- and AT-2-specific primers. In PAZ-6 cells, 1 µM L-755.507 and 1 nM T₃ induced UCP3 mRNA expression 1.7- and 1.4-fold (n = 4, p < 0.05), respectively, in comparison to basal conditions. The AT-1- and AT-2-specific primers, which failed to produce PCR products in myocyte cDNA, resulted in the generation of PCR products. Results with L-755.507 were verified by RNase protection assay showing only fragments specific for AT-1 and AT-2 (Fig. 3A).

UCP3 Promoter Sequences Are Highly Divergent between Humans and Rodents-- Multiple sequence alignment of human, rat, and mouse (GenBank^TM accession number AB011070) UCP3 promoter-exon 1 regions revealed a striking degree of homology between rat and mouse UCP3 promoters (Fig. 4A). Overall sequence identity along 2685 nt of mouse promoter-exon 1 sequence was ~83% with only 6% gaps necessary to achieve optimized alignment. Comparison of both rodent promoters with the human UCP3 promoter showed an average identity of ~53% with more than 22% gaps introduced to achieve optimal alignment results. BLAST searches revealed several regions with higher than average sequence homology. The high homology that was observed in the proximal promoter-exon 1 region ended abruptly ~390 nucleotides upstream of the human exon 1-intron 1 boundary with the insertion of a composite MIR1/Alu half-site element in the human UCP3 promoter (52, 53). Two additional highly conserved blocks in human, rat, and mouse UCP3 promoters were found to represent ancient repeats inserted into the genome at orthologous sites before the mammalian radiation. The L1MB7 repeat, located adjacent to the disruptive MIR1/Alu composite sequence, is a member of the medium reiteration frequency sequences and belongs to a subfamily of ancestral mammalian-wide LINE-1 repetitive elements (54). The other highly conserved sequence was identified to represent MIR1, a member of the group of mammalian-wide interspersed repeats (MIRs) (52). The L1MB7 and MIR1 sequence blocks flanked a total of five human EST clones (GenBank^TM accession numbers AI902389, AI902390, AA421836, AA625416,and AI198075) that were identified by BLAST searches. The location of the five EST clones corresponded roughly to three higher than average similarity values among human, rat, and mouse UCP3 promoters suggesting the existence of homologous rodent genes in this region (Fig. 4A).

A rootless phylogenetic tree based on the multiple sequence alignment data and calculated according to the Neighbor-joining method (37) is shown in Fig. 4B. Mouse and rat sequences exhibited nearly the same distance from a putative common ancestor sequence with a nucleotide substitution rate <10% per site, whereas the human UCP3 promoter-exon 1 region was highly divergent displaying a nucleotide substitution rate of >30% per site. The substitution rate increased to >40% after exclusion of the conserved elements L1MB7 and MIR1. Results from phylogenetic analyses were nearly identical when performed with or without corrections made for gaps and multiple nucleotide exchanges according to Kimura (38) (data not shown).

In conclusion, these studies contain novel information on human UCP3 gene structure and regulation. The human UCP3 gene is expressed in skeletal muscle and brown adipocytes. Rates of synthesis and/or degradation of UCP3 mRNA isoforms differ between human adipose and muscle tissues. Moreover, UCP3 mRNA expression in human adipose and muscle tissue is regulated by tissue-specific factors as demonstrated by tissue-specific transcript initiation. These results raise the exciting possibility that UCP3 gene expression in skeletal muscle and adipose tissue can be selectively regulated for therapeutic purposes. The divergence of human and rodent UCP3 regulatory regions suggests functional diversification of human UCP3 gene regulation. These novel findings deserve consideration, when results from animal experiments are extrapolated to human UCP3 physiology.

	ACKNOWLEDGEMENT

We thank Michael H. Fisher (Merck) for providing a sample of the 3 agonist L-755.507.

	FOOTNOTES

^* This work was supported by a grant from the Medizinische Forschungsgesellschaft Salzburg and the Stiftung Propter Homines, Vaduz, Liechtenstein.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF050113 and AF168989.

To whom correspondence should be addressed: Dept. of Laboratory Medicine, Landeskliniken Salzburg, A-5020 Salzburg, Austria. Tel.: 43-662-4482-3800; Fax: 43-662-4482-885; E-mail: w.patsch@lks.at.

Published, JBC Papers in Press, August 24, 2000, DOI 10.1074/jbc.M005713200

	ABBREVIATIONS

The abbreviations used are: UCP3, uncoupling protein-3; BAT, brown adipose tissue; T₃, triiodothyronine; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; H, human; MR, mouse/rat; DMEM, Dulbecco's modified Eagle's medium; nt, nucleotide; MIRs, mammalian-wide interspersed repeats; EST, expressed sequence tag.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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