The Guanosine Monophosphate Reductase Gene Is Conserved in Rats and Its Expression Increases Rapidly in Brown Adipose Tissue during Cold Exposure*

Non-shivering thermogenesis is required for survival of rodents during cold stress. Uncoupling protein-1 acts in brown adipose tissue (BAT) to transport protons, thus dissipating the proton gradient across the inner mitochondrial membrane. This permits respiration uncoupled from ATP synthesis. UCP-1 function is inhibited by the binding of purine nucleotides, with GTP/GDP being more potent than ATP/ADP. We used a cDNA subtraction analysis to identify cDNAs rapidly induced by cold exposure. One of these encodes rat guanosine monophosphate reductase (GMP-r). This was surprising in that previous data had suggested that this enzyme was absent in rodents. Rat GMP-r is 96% identical to human GMP-r, and its mRNA is increased 30-fold in BAT within 6 h of cold exposure. The gene is also expressed (but not cold-responsive) in muscle and kidney, but not in white fat. We speculate that the physiological function of the marked increase in BAT GMP-r during cold stress may be to deplete the brown adipocyte of guanine nucleotides, converting them to IMP, thus permitting enhanced UCP-1 function. This is a previously unrecognized regulatory aspect of thermogenesis, an essential physiological response of rodents to cold.

Adaptation to cold in rodents requires a physiological process called non-shivering or facultative thermogenesis. Brown adipose tissue (BAT) 1 plays the dominant role in this response through its capacity to produce heat by uncoupling oxidative phosphorylation. Thermogenesis in BAT is initiated in the hypothalamus and is effected via the intense sympathetic nervous innervation of this tissue. Many genes are, either directly or indirectly, transcriptionally up-regulated by the adrenergic stimuli initiated by cold exposure. Norepinephrine activates lipolysis in BAT and rapidly increases the synthesis of uncoupling protein (UCP-1) (reviewed in Ref. 1). The free fatty acids can then act to uncouple respiration from oxidative phosphorylation. UCP-1 is a proton translocator, highly and exclusively expressed in brown fat cells, which dissipates the proton gradient across the inner mitochondrial membrane (for review see Ref. 2). Recent data suggest that the role of UCP-1 in H ϩ transport is either to transport the fatty acids or the anion OH Ϫ , which can act as cycling protonophores therefore reducing the proton gradient across the inner mitochondrial membrane (3)(4)(5)(6)(7)(8). The H ϩ transport process is tightly regulated in that it is allosterically inhibited by the binding of endogenous purine nucleotides to a specific binding site in UCP-1 (2). The affinity of these nucleotides for UCP-1 binding decreases in the order GTP Ͼ GDP Ͼ ADP (9).
To identify genes involved in facultative thermogenesis we performed a cDNA subtraction analysis to identify BAT mRNAs that increase during acute cold exposure (10). We isolated and cloned one such cDNA that encodes the guanosine monophosphate reductase (GMP-r). The mRNA encoding this enzyme increases 30-fold within 6 h of exposure to 4°C. GMP reductase catalyzes the following reaction: REACTION 1 Thus, this enzyme catalyzes the conversion of guanine nucleotides to IMP, which can then be redirected to AMP synthesis. This is the first identification of a rodent cDNA encoding this protein. In fact, several previous reports concluded that GMP reductase, present in Escherichia coli and humans, was lost during the evolution of rodents and that an unrelated enzyme catalyzes this reaction in those species (11,12). The present studies show that this is not the case and demonstrate, furthermore, a marked and rapid increase in GMP reductase in BAT after cold exposure suggesting a previously unrecognized and critical role for the reaction catalyzed by this enzyme in non-shivering thermogenesis. As mentioned, guanine nucleotides are more potent inhibitors than are adenine nucleotides of fatty acid transport by UCP-1 (8,9,13). We speculate that GMP reductase may enhance UCP-1 function by reducing its inhibition by endogenous guanine nucleotides.

EXPERIMENTAL PROCEDURES
Cold Exposure and RNA Isolation-Male Sprague-Dawley rats (100 -125 g) were obtained from Zivic-Miller Laboratories and used under a protocol approved by the Animal Use Committee at Harvard Medical School. Rats were divided into two groups: one (control group) was kept at 21°C (cDNA(Ϫ)), and the other was maintained at 4°C for 5 h (cDNA(ϩ)). Rats were killed, and interscapular BAT was dissected and immediately frozen in liquid N 2 . Total RNA was isolated by the guanidinium/phenol method (14). Poly(A) ϩ RNA was selected by passage through an oligo(dT) column (Amersham Pharmacia Biotech, Type 7).
Subtraction Library and cDNA Cloning-A PCR-based subtractive hybridization strategy was followed to isolate cDNAs that were rapidly increased during cold exposure (10). The two mRNA populations were converted to double-stranded cDNAs, fragmented by digestion with * This work was supported by Research Grants DK36256 and DK07529. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 1 The abbreviations used are: BAT, brown adipose tissue; GMP-r, GMP reductase; UCP, uncoupler protein; PCR, polymerase chain reaction; bp, base pair(s); MOPS, 4-morpholinepropanesulfonic acid.
RsaI and AluI restriction enzymes, and ligated to a double-stranded phosphorylated oligodeoxynucleotide linker, which had one blunt end and one 4-base 3Ј-protruding end (CTCTTGCTTGAATTCGGACTA and TAGTCCGAATTCAAGCAAGAGCACA) to allow several PCR amplifications of the linker-ligated cDNA fragments (10). The amplified cDNA(Ϫ) and cDNA(ϩ) fragments were the starting material for subtractive hybridization. Biotinylated driver cDNA(Ϫ) and nonbiotinylated tracer cDNA(ϩ) were mixed, precipitated, and redissolved, and the process was repeated six times to enrich the cDNA(ϩ) pool in cDNAs encoding stimulated transcripts. Following this, the enriched cDNA plasmid library was digested with EcoRI, which cuts in the adapter oligo, and subcloned into pBS-KS (Stratagene). Screening of the colonies was performed using the same enriched cDNA as a probe, and 25 highly positive clones corresponding to the most representative cDNAs were picked for small scale preparation of plasmid DNA. The inserts were labeled and isolated and then used to probe mRNA dotblots from stimulated and nonstimulated BAT, liver, kidney, and white adipose tissue. This allowed identification of cDNAs hybridizing to mRNAs that were expressed only in BAT and that increased markedly during acute cold exposure. One such cDNA fragment was 320 bp in length. It was sequenced and found to be highly similar to the human GMP reductase cDNA. This was then used as a probe to screen a Lambda Zap II cDNA library prepared from BAT mRNA from cold exposed rats.
BAT cDNA Library Construction and Screening-Poly(A) ϩ RNA was prepared from BAT exposed to 4°C for 48 h and used for the synthesis of cDNA using a cDNA synthesis kit from Stratagene. The size-fractionated cDNAs (greater than 0.8 kilobase pair) were ligated into Lambda Zap II phage vector, packaged, transfected into SURE cells, plated, transferred onto nylon filters (GeneScreen Plus, NEN Life Science Products), and screened using the cDNA probe derived from the subtraction screen using standard techniques. Three positive clones were plaque-purified and converted to Bluescript SK plasmid by in vivo excision as described in the Stratagene protocol. Two of these contained approximately 1.4-kilobase pair inserts. One of these (corresponding to the full-length rat GMP reductase cDNA) was selected for DNA sequencing.
Northern Blot Hybridization-Total RNA (20 g) was denatured and electrophoresed on a 2.2 M formaldeyde, 1% agarose gel in 1ϫ MOPS buffer and transferred to nylon membranes (GeneScreen) as described previously (14). The EcoRI 1452-bp fragment corresponding to the fulllength GMP reductase cDNA was labeled with [ 32 P]dCTP using random primers (Prime It kit, Stratagene). Filters were hybridized for 18 h at 44°C (50% formamide, 5ϫ SSC, 2ϫ Denhardt's solution, 0.1% SDS, and 10 mg/ml salmon sperm DNA). The last wash was 0.1% SSC, 0.1% SDS at 44°C for 20 min. Autoradiograms were obtained from the filters and quantified by computer-assisted laser densitometry (Molecular Dynamics, Sunnyvale, CA). For Type 2 deiodinase expression, a cDNA 846 bp long corresponding to the coding sequence of the rat D2 cDNA (rD2) was labeled as described before. The rD2 cDNA was a generous gift of Dr. Donald St. Germain (15).
In Vivo Studies-Male rats (about 120 g) were kept at room temperature (controls) or in the cold room (ϩ4°C) from 0 to 48 h. All animals were subjected to a light-dark cycle (6 h light, 18 h dark) and had free access to food pellets and water. Animals were thyroidectomized by the supplier with parathyroid reimplantation and were killed 10 days after surgery. Some animals were made hyperthyroid by daily intraperitoneal injections of 3,5,3Ј-triiodothyronine, 50 g/100 g every other day for 7 days, and normal animals of the same age and supplier were used as controls. Trunk blood was collected for thyroxine measurements to confirm thyroid status.
Western Blot Analysis-Analysis of the GMP reductase protein expression in BAT and spleen was performed by Western blotting using 50 g of homogenate protein. Pooled tissue from two animals for each point was ground in liquid N 2 , and protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad). Total protein was boiled in SDS sample buffer (62.5 mM Tris (pH 6.8), 10% glycerol, 7.5% SDS, and 6% ␤-mercaptoethanol and run on a 10% polyacrylamide gel). Proteins were electrophoretically transferred to nylon membrane (Immobilon-P) by standard procedures. The membranes were stained with Ponceau S (Sigma) to check for equal loading and efficiency of transfer. Polyclonal antibodies against the amino terminus of human GMP reductase (residues 2-16) were kindly provided by Dr. Ernest Beutler (San Diego, CA) and used as described previously at a dilution of 1/3000 (16). Proteins were detected by enhanced chemiluminescence using the ECL system (Amersham Pharmacia Biotech) with horseradish peroxidase-conjugated second antibodies (Bio-Rad) at 1/4000 dilution.

RESULTS
Cloning and Sequence Analysis of Rat GMP Reductase cDNA-To identify genes involved in cold adaptation in brown adipose tissue, a PCR-based subtractive cDNA library was performed using interscapular BAT poly(A) ϩ from control and cold-stimulated rats. One of the cDNA fragments (clone 4) derived from the cDNA(ϩ) that met our selection criteria (expressed exclusively in BAT and markedly increased during acute cold exposure) was subcloned and sequenced. The sequence obtained was highly similar to the human GMP reductase, and the partial cDNA was used to screen a cDNA library prepared from BAT from cold-exposed rats (see "Experimental Procedures"). A 1452-bp clone was identified and sequenced (Fig. 1). It contained an open reading frame of 1035 nucleotides, encoding a deduced protein of 345 amino acids that is 96% identical to the human GMP reductase (Fig. 2). The two cDNAs share a 79% identity at the nucleotide level. There is a polyadenylation site (AATATA) in the cDNA located at nucleotide 1492, 14 nucleotides upstream of the poly(A) tail. On the basis of this high homology, this cDNA encodes the rat GMP reductase.
Tissue Expression of the GMP Reductase Gene in the Rat-The identification of the GMP reductase cDNA prompted us to investigate factors regulating its expression in greater detail. To explore its expression during cold exposure, we analyzed nine rat tissues by Northern blot analysis comparing the GMP reductase expression of control and cold-stimulated rats. As shown in Fig. 3, the GMP reductase mRNA is highly expressed in BAT as compared with other tissues. It is also expressed in skeletal and cardiac muscle, but in those tissues its expression is 5-fold lower than in unstimulated BAT, and it does not increase during cold stress. GMP reductase is not expressed in testis, lung, liver, or spleen, but it is weakly expressed in kidney from cold-stimulated animals. In contrast to the high levels of GMP reductase mRNA in brown fat, no specific signal could be detected in white fat (Fig. 3).
Effect of Cold Exposure on GMP Reductase mRNA and Protein-Exposure to 4°C for 6 h caused a marked increase in GMP reductase mRNA only in BAT. To analyze this issue further we compared GMP reductase mRNA and protein expression after increasing the time of cold exposure. After only 3 h at 4°C, GMP-reductase mRNA increased 10-fold reaching the maximum levels of 30-fold after 6 h (Fig. 4A). The mRNA remained elevated during 4 days at 4°C. The increases in GMP reductase mRNA are similar in timing to those of D2 mRNA (Fig. 4B), the enzyme that generates 3,5,3Ј-triiodothyronine in BAT. Actin mRNA did not change significantly in the same samples (Fig. 4C).
Western blotting with an antiserum directed against residues 2 to 16 of the human GMP reductase (100% identical in the rat protein (Fig. 2)) showed a 37-kDa protein in BAT lysates that is not visible in spleen (Fig. 5A). After 6 h of cold exposure the 37-kDa band is increased 4-fold over control and reaches its maximum level at 24 h (7-fold). It is still 3-fold higher than control after 72 h of cold exposure (Fig. 5B).
Effect of Thyroid Status on GMP Reductase in BAT-Thyroid hormone level influences adrenergic status and lipogenesis in rat BAT with increased norepinephrine turnover in hypothyroid animals and reduced sympathetic tone during hyperthyroidism (reviewed in Refs. 1 and 17). Interestingly, thyroid status strongly affected GMP reductase expression at both the mRNA and protein levels. As shown in Fig. 3, GMP reductase mRNA is 4-fold higher in BAT from hypothyroid rats maintained at 21°C than in controls. On the other hand, BAT GMP reductase mRNA is reduced 3-fold in hyperthyroid rats. In thyroidectomized animals, GMP reductase protein is also in-creased 3-fold compared with euthyroid rats, and conversely, it is suppressed to 30% of control in BAT from hyperthyroid animals (Fig. 5B). DISCUSSION In rodents, cold adaptation is essential for survival. During the first hours of cold exposure there is a marked stimulation of BAT through the sympathetic nervous system culminating in thermogenesis in this organ (2). We have used a PCR subtraction strategy to identify a previously unrecognized gene involved in these early events. After only 3 h of cold exposure, we detect a marked increase in the expression of GMP reductase mRNA and protein. This enzyme reduces GMP, producing IMP, thus representing the only step by which guanine nucleotides can be converted to IMP and subsequently to adenine nucleotides.
The human GMP reductase enzyme has an unusual history. It was initially cloned as one portion of a cDNA cloning artifact and thought to represent part of a post-transcriptionally generated chimeric protein (11,18). This error was recognized subsequently (16,19,20), but since then, no further studies The filter was first hybridized with an 0.8-kilobase pair probe from the rat GMP reductase cDNA. Thereafter, a second hybridization was performed on the same filter with a type 2 deiodinase-specific probe (B). C shows results of probing with rat actin, and D shows the ethidium bromidestained gel prior to transfer.
FIG. 5. Western blot analysis of GMP reductase protein expression in BAT and spleen from rats exposed to 4°C for 5 h (A) and various times (B). Tissue homogenate protein (50 g) from control BAT and from cold-stimulated rats were examined by Western blot analysis using antibody directed against residues 2-16 of GMP reductase (B) as described previously. Equal protein loading and transfer were verified by Ponceau staining of the membrane. In the last two lanes of panel B, BAT protein from rats 10 days after thyroidectomy (Tx) or after treatment with 3,5,3Ј-triiodothyronine (T3) for 7 days (see "Experimental Procedures") was similarly analyzed. have been performed on this gene besides the characterization of its structure and genetic polymorphism and its assignment to human chromosome 6 (12,21,22). Previous evidence suggested that this enzyme, present in E. coli and humans, was somehow lost during evolution in rodents. The failure to detect a similar cDNA in the mouse and hamster genomic DNA (21), together with earlier data showing that rat tissue can convert guanine to hypoxanthine (23), had previously suggested that an unrelated or distantly related enzyme could catalyze this reaction in these species (20). Here we show that this is not the case since not only is GMP reductase expressed in rat BAT, but its expression is highly regulated in this tissue. Rat GMP reductase is highly homologous to human GMP reductase, sharing over 95% identity at the amino acid level.
The tissue distribution of GMP reductase has not been explored previously. Rat GMP reductase mRNA is highly expressed in BAT, even under basal conditions, and is also present in skeletal and cardiac muscle and kidney, but is absent in testis, liver, lung, spleen, and interestingly, white fat. Furthermore, in BAT, its expression is responsive to conditions that regulate BAT function such as cold stimulation or thyroid status (Figs. 3 and 4). The presence of GMP reductase mRNA in other tissues implies more general functions of this enzyme outside those of thermogenesis. However, after only 3 h of cold exposure, BAT GMP reductase RNA increases 10 times over control, reaching the maximum expression level of 30-fold after 6 h (Fig. 4). Previous work clearly demonstrated that optimal expression of the UCP-1 gene during cold stress requires both norepinephrine and 3,5,3Ј-triiodothyronine (24 -27). The function of GMP reductase in cold-stimulated BAT has not been explored. Its rapid and marked increase during cold exposure suggests that high levels of this enzyme are critical in those cells.
How can a requirement for GMP reductase in the acute response of brown adipocyte be explained? It can be hypothesized that the nucleotide binding site of UCP-1 is normally blocked primarily by guanine, rather than adenine nucleotides, due both to a higher affinity of the guanine nucleotides and as a high ratio of guanine to adenine nucleotides in the mitochondrial intermembrane space. In this scenario, GMP reductase will facilitate UCP-1 function by releasing endogenous inhibition by these nucleotides. This may be critical in this process since free fatty acids per se do not displace purine nucleotides that act as allosteric inhibitors of UCP-1 (3). Other effects of GMP reductase may also play a role in the brown fat response. The reductive deamination of GMP to IMP could potentially increase the pool of adenylosuccinate, the precursor of AMP, thus making this nucleotide available for the eventual formation of the second messenger, cAMP, crucial for norepinephrine stimulation of BAT thermogenesis.
The parallel effects of cold exposure to increase UCP-1, D2, and GMP reductase mRNAs (Figs. 3-5) point to overlapping and synergistic mechanisms that have evolved for stimulation of heat production in rodents. Presumably, these genes will be found to contain common transcriptional control elements, such as cyclic AMP response elements, to explain their similar response. The fact that GMP reductase, like D2, is increased in BAT of hypothyroid rats and decreased in hyperthyroidism (Figs. 5) shows that compensatory changes occur in the expression of both genes to modulate facultative thermogenesis in a physiologically appropriate fashion.
The cloning of the GMP reductase cDNA, the identification of its tissue distribution, the demonstration of its regulation by cold exposure, and thyroid status have revealed new insights into the role of purine interconversion in BAT physiology. Nonshivering thermogenesis is an important mechanism for dissipating energy and hence controlling body weight. Correlation of GMP reductase with BAT heat production makes it a candidate gene potentially related to human obesity and related disorders.