Expression of Uncoupling Protein mRNA in Thermogenic or Weakly ...

THEJOURNAL

OF

BIOLOGICAL CHEMISTRY

Vol. 261. No. 30, Issue of October 25. pp. 13905-13910,1986 Printed in U.S.A.

D 1986 by The American Society of Biological Chemists, Inc.

Expression of Uncoupling Protein mRNA in Thermogenic or Weakly Thermogenic Brown Adipose Tissue EVIDENCEFOR A RAPID0-ADRENORECEPTOR-MEDIATED STEP DURINGACTIVATION OF THERMOGENESIS*

AND TRANSCRIPTIONALLYREGULATED

(Received for publication, April 28, 1986)

Daniel Ricquierzg, Frederic Bouillaud$, Philippe Toumelinz, Gerard Moryll, Raymond Bazin(1, Jonathan Arch**, and Luc Penicaudll From the $Centre de Recherches sur la Nutrition (Centre National de l a Recherche Scientifiqw, LP1511), 9, rue Jules Hetzel, F-92190 Meudon, France, the llUniversites Pierre et Marie Curie et Paris VII (Centre National de Ia Recherche Scientifique, UA307), F- 75230 Paris, France, the lllnstitut National de la Sante et de la Recherche Medicale, U177, F- 75006 Paris, France, and the **Beechum Pharmaceutical Research Division, Epsom, United Kingdom

(1). It is not synthesized asa larger M,precursor, contrary to most of the mitochondrial proteins(6-12), and exhibits quantitative adaptive changes (1-3, 5, 13). Thermogenesis in the brawn fat of lean mammals can be turned on either during exposure to cold (1-5) or toa lesserextent duringhyperphagia (14). These stimuli cause cell hyperplasia, increase mitochondriogenesis, and induce a high level of UCP (1-3, 5). In contrast, brown adipose tissue of obese animals often has a poor thermogenic capacity, and this could be related toa low amount of UCP (15,16). Cell-free synthesis experiments have showna 5-fold increase in the synthesisof UCP observed with mRNA isolated from brownadipose tissue of rats maintained at 5 “C for several days (6, 8, 10). Moreover, experiments based on in vitro synthesis and immunoprecipitation have demonstrated a striking induction in the mRNA encoding the UCP of both animals having a pheochromocytoma tumor or subjected to norepinephrine infusion (8).These data, together with data from other studies (13, 17-20), suggest that UCP synthesis is under noradrenergic control. Recently, we have cloned the (21). DNA sequences complementarytoratUCPmRNA Using this probe a remarkable increase in the level of the UCP mRNA canbe detected during cold exposure after only 1 h (22). A similar effect occurs when the animal is infused with norepinephrine (21) or subjected to a “cafeteria” diet (23). Jacobson et al. (24) have recently isolated clones containing mouse UCP cDNA. They detected a 4.5- and 10-fold UCP nlRNA induction inmice exposed to 5 “C for 2 and 6 h, Brown adipose tissuemitochondria possess as a major respectively. component of theirinnermembrane a highly specialized Here a cDNA has been used to probe UCP mRNA levels in uncoupling protein (UCP’) which is unique to this tissue(1- physiological (exposure of adults to thecold, birth) or patho5 ) . It has the ability to short-circuit the proton electrochem-logical (genetic or surgical obesity) situations. In addition the ical gradient generated across this membrane during substrate in vivo effects of BRL 2683014 on the induction of the UCP oxidation, thus promoting energy dissipation as heat (1). One mRNA have been studied since Arch et al. (19, 20, 25) have of the major characteristicsof the protein is itshigh affinity shown this compound to cause a selective activation of the binding site for nucleotides such as GDP, GTP, ADP, ATP or atypical P-adrenoreceptor in this tissue with consequent stimulatory effect on respiration,lipolysis, and UCPlevel of brown * This work was supported by grants from the Centre National de adipocytes. Moreover, the elongation of nascent RNA chains la Recherche Scientifique, Institut de la Sante et de la Recherche possible Mdicale, and Direction des Recherches Etudes et Techniques. A in isolatednuclei hasbeen used toexaminethe part of this data was presented to the614th Meeting of the Biochem- transcriptional regulation of Ucp gene. We report here that or inhibiical Society held in Oxford on July16-l8th, 1985. The costs of UCP mRNA synthesis is altered during activation publication of this article were defrayed in part by the payment of tion of thermogenesis. Such changes are rapidly controlled by page charges. This article must therefore be hereby marked “aduer- P-adrenoreceptors and transcription of the Ucp gene.

A cloned cDNA sequence for the unique mitochondrial uncoupling protein of rat brown adipose tissue has been used to assay the corresponding mRNA in several situations. When thermogenesis in brown adipose tissue is stimulated(exposure of adult rats to the cold, birth) a rapid andprolonged increase in thelevel of uncoupling protein mRNA is observed. Such an increase canbe mimicked by injection of animals witha new B-adrenoreceptor agonist BRL 26830A. Conversely it isknown that mice and rats with genetic or surgical obesity have a weakly thermogenic brown adipose tissue with a reduced norepinephrine turnover. A reduced level of uncoupling protein mRNA was measured in obese fa/fa rats 10 days or 10 weeks old and in obese rats with a lesion of the ventromedial hypothalamic area but not in obese obtob mice. Moreover, exposureof obese animals to cold or dosing with BRL 26830A strikingly increased thelevel of uncoupling proteinmRNA. Measurement of the relativeconcentration of nascent Ucp transcripts innuclei isolated from brown adipose tissue indicates that Ucp gene is acutely (within 15 min) regulated at the level of transcription and iscontrolled via activation of B-adrenoreceptors of plasma membrane. Ucp gene transcription is decreased in obese fatfa rats but can be fully and rapidly turnedon after injection of BRL 26830A.

tisernent” in accordance with 18U.S.C.Section 1734 solely to indicate this fact. 3 To whom correspondence should be addressed. The abbreviations used are: UCP, uncoupling protein; Ucp gene, gene of uncoupling protein; VMH, ventromedial-hypothalamic.

EXPERIMENTALPROCEDURES

ChemZ~als-[cu-~*P]dCTP,[w3*P]TTP, and [a-”P]UTP were obtained from the Amersham International Radiochemical Centre.

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UCP mRNA Expression Brown in

Oligo(dT)-cellulose was obtained from Collaborative Research (Waltham, MA) or from Boehringer Mannheim; messenger-activated paper was obtained from Orgenics (Yavne). Poly(T) strands were obtained from Pharmacia (Uppsala) and BRL 26830A from Beecham Laboratories (Epsom). Deoxyribonuclease I, DNA polymerase I, and RNase A were obtained from Boehringer Mannheim or from Appligene (Illkirch). Animals-Lean male Wistar rats (200 g, body weight) were either exposed to cold or injected with a 8-adrenoreceptor agonist. Rats exposed a t 5 “C were singly caged. The 8-agonist BRL 26830A was solubilized in water and injected intraperitoneally a t a dose of 10 mg/ kg of body weight. Rats so treated for 48 h received the drug a t 0-18 and 36 h. Rats were either obese males (fa/fa) and lean littermates (+/?) of the Zucker strain fa/fa aged 10 days or 8 or 10 weeks, or 12week-old female Wistar rats made obese by lesion of the ventromedial hypothalamus nuclei (VMH).Bilateral VMH lesions were made under Ketalar anesthesia (80 mg/kg, intraperitoneal) as described previously (26). The lesion was considered as successful when the weight gained within 7 days was greater than 70g. Interscapular brown adipose tissue was excised 3 weeks after lesions. 10-day-old fa/ fa rats were identified according to theprocedure previously described (27). C57B1 genetically obese ob/ob mice and their lean littermates were also studied with the @-agonistBRL 26830A. For developmental studies, animalswere killed at theindicated age. Isolation of Brown Fat RNA and Poly(A)+ RNA-Isolation of total RNA was carried out as previously described using the phenol procedure (6, 8, 21). The yield of extraction was reduced by a factor 2 when the thiocyanate guanidium method was used. Poly(A)+ RNA was isolated either using 2 passages on oligo-dT affinity column or using paper-affinity chromatography as previously described (6,8,21, 22). Hybridization of UCP cDNA Probe to RNA Fixed to Nitrocellulose Membrane-Both “Northern” and“dot blot” methods were employed. For the former method RNA was previously electrophoresed in agarose gel containing formaldehyde as described in Ref.21. Cloned pUCP 36 insert (21) was labeled with 32Pand used to probe the RNA; poly(T) strands were also labeled with 32Pand used to probe the poly(A) tail of RNA bound to nitrocellulose. This procedure was used to estimate theamount of poly(A)+RNA hybridized to nitrocellulose filters. Hybridization and washing conditions of these filters were as described previously (21). Validity of UCP mRNA variations was also verified by hybridization of brown fat RNA to mice p41 cellular actin (28), chicken pCB12 calmodulin (29), or A,Cs rat aldolase B (30) probes. RNA Synthesis in Isolnted Nuclei-Nuclei were purified for in vitro transcription experiments essentially according to Schibler et al. (31). Each elongation reaction was performed with nuclei isolated from the pooled tissue of 6 animals. The in vitro elongation reactions (30 “C for 15 min) were made as described in Ref.32. The reaction was terminated by the addition of DNase I made RNase free by treatment with proteinase K inthe presence of CaC12(33). RNA wereextracted, ethanol precipitated, and dissolved in the hybridization mixture. After counting of radioactivity into RNAs, labeled transcripts (IO6 cpm) were hybridized to recombined pBR plasmids (12 pg) immobilized on “Hybond N (Amersham Corp.) 5-mm diameter disksor sheet filters. Hybridization (42 “C for 60 h) andwashing of filters including RNase treatment were carried out essentially as described in Ref. 32.

Adipose Tissue

Brown Adipose Tissue-Fig. 1 shows that the UCP mRNA level can be significantly increased after 1-hexposure of rats at 5 “C. This induction is continuous and increased during prolonged cold exposure, a maximum level of UCP mRNA being detected after 48 h. Densitometric scanning of nonsaturated autoradiography indicates that the UCP mRNA level is increased by a factor of2.5, 4, and 9 at 1, 5, or 48 h, respectively. In other experiments we have observed an increased UCP mRNA level in rats exposed at 5 “C for 30 min or less (not shown). Such a rapid induction suggested an effect at the transcriptionallevel. UCP mRNA during Development of Rats-UCP mRNA level has been measured in end-termfetuses and in developing animals. UCP mRNA was detected in fetuses and was strikingly increased during the hours following birth (Fig. 2). A high level was observed in 3-day-old animals, but a reduction occurred in animals during aging. The data agree with GDPbinding titration of UCP during development (35). The large increase observed at birth probably results from cold shock and corresponds to the starting point of development of the thermoregulatory system. Moreover, these data give prominence to a difference between rat and rabbit since in this latter species cell-free synthesis and immunoprecipitation experiments have shown an increase in UCP mRNA during intrauterine life but not at birth (9,lO). P-Adrenoreceptor Control of UCP mRNA Synthesis-Preliminary support for P-adrenoreceptor control of UCP mRNA synthesis was provided by treatment of animals with propranolol whereupon the increase in UCP mRNA usually observed after 1 h of cold exposure was inhibited (22). However, propranolol effects are not always entirely mediated via P-adrenoreceptors. Recently, a new P-adrenoreceptor agonist has been presented (25) which has a selective action at the P-adrenoreceptor that mediates lipolysis and respiration in adipocytes

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RESULTSANDDISCUSSION

The cloned cDNA used in this study is specific for brown adipose tissue UCP (21). The identity of this cDNA has been confirmed by nucleotide sequencing (12), and the cDNAderived amino acid sequence of rat UCP (12) is in excellent agreement with the protein-derived sequence of hamster UCP (34). In addition to rat mRNA, pUCP 36 cDNA hybridizes to UCP mRNA from mouse (Ref. 21 and this study), hamster (21), and to a lesser extent that of rabbit, guinea pig, and man.’ Using the “Northern blot” procedure rat UCP cDNA detects two RNA bands, a major one at 15 S (1.5 kilobase pairs) and a minor one at 18s (1.9 kilobase pairs) (Figs. 1-3 and 5-7). Two RNA signals have also been detected in mouse when using cloned mouse UCP cDNA (24). Increase in UCP mRNA Level during Acute Activation of

* D. Ricquier and F. Bouillaud, unpublished data.

FIG. 1. Increase in UCP mRNA level after stimulation of brown adipose tissue by exposure of rats to cold; Northern analysis. Poly(A)+ RNA was isolated using chromatography on oligo(dT)-cellulose column. Each lane corresponds to 1 pg of RNA. RNA was hybridized to the32P-labeledpUCP 36 cDNAinsert. Lunes 1-4, poly(A)+ from brown adipose tissue of rats either kept a t room temperature ( l a n e I ) or exposed a t 5 “C for 1 h ( l a n e 2), 5 h (lane 3 ) , or 48 h ( l a n e 4 ) . Lune 5, poly(A)+ from rat liver. Poly(A)+ RNA recovery was normalized by hybridization to a 32P-labeledpoly(T) probe. Moreover, no large alteration was observed when these RNAs were hybridized to p41 cellular actin cDNA (not shown).

UCP mRNA Expression in Brown Adipose Tissue 1 2 3

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FIG.3. Effect of a new B-adrenoreceptor agonist (BRL 26830A) on UCP mRNA level; comparison with the effect of cold exposure; Northern analysis of UCP mRNA. Left, total RNA. Lunes 1 and 4 , animals maintained a t 25 "C; lunes 2 and 5, animals exposed a t 5 "C for 1 h (lune 2 ) and for 48 h (lune 5);lunes 3 FIG.2. Assay of UCP mRNA level in brown adipose tissue and 6, animals maintained a t 25 "C and dosed with BRL 26830A for of rats during development. Left, Northern analysis. 1 pg of 1h (lune 3 ) or 48 h (lune 6 ) .Similar datawere obtained withpoly(A)+ RNA. The upper arrow corresponds to the18 S value. Right, poly(A)' poly(A)+ RNAwas electrophoresed in each lanebefore hybridization to "P-labeled UCP probe. Lane I , brown fat RNA from end-term rat RNA. Lune I , control animals; lune 2, rats dosed with BRL 26830A for 1 h; lune 3, rats dosed for 2 h. fetuses (21.5 days postcoitum); lune 2, RNA from newborn rat 6-10 h old; lunes 3-5, rats aged 3, 10, and 20 days, respectively. Right, dotblot analysis. Equal amounts of poly(A)+ RNA were dotted on nitroo b/o b lean cellulose and hybridized either to"P-labeled poly(T) probe or to '*Plabeled UCP probe. I , end-term fetuses; 2, newborn rats; 3,3-day-o1d rats; 4, 10-day-old rats; 5, 20-day-old rats. A striking UCP mRNA increase was detected a t birth. The specificity of this increase was also verified by hybridization of RNAs topCB12 calmodulin and p41 cellular actin probes (not shown). (20, 25). Figs. 3, 4, and 6show thatacute or prolonged treatment, or both, of rats ormice with BRL26830A is capable of increasing strongly the UCPmRNA level. These data are in excellent agreement with studies where an increased level of UCP was recordedin animals chronicallyinjectedwith BRL 26830A (19, 20) or with norepinephrine (18). Data presented indicate that UCP synthesis is a p-adrenoreceptormediated process. It could thus be postulated that Ucp gene transcription is regulated by signalsgeneratedduringthe action of norepinephrine released by nerve endings at the surface of brown adipocytes. UCP mRNA in Obese oblob Mice-UCP mRNA was assayed in brown adipose tissue of genetically obese ob/ob mice. In a first series of experiments a lower level of UCP mRNA in tissue of 10-week-old obese mice was detected, but in a second and large series of experiments, an almost normal (or slightly reduced) level was observed (Fig. 4) as recently reported (24).We are unable toclearly explain this discrepancy. Moreover, conflicting data concerning UCP level had been reported by several groups. Authors have described a reduced level of UCP in mitochondria (36,37) while others measured 9 normal concentration (38, 39). When the BRL 26830A pagonist compound was given to obese ob/ob mice a significant and normal increase was observed (Fig. 4). This supports the results of Jacobson et al. (24) who observed an increase in UCP mRNA in cold-exposed obese ob/ob and db/db mice which approximated the increase in control animals. The pagonist-stimulated increase in UCP mRNA observed here in

FIG.4. Assay of UCP mRNA level in brown adipose tissue of genetically obese ob/ob mice; effect of BRL 26830A injecweeks old. Dots tion; dot-blot hybridization. Animalswere10 correspond to 1,2, or4 pg of RNA. Lean and obese ob/ob mice were dosed either with water (line a ) or with BRL 26830A for 1 h (line b ) or 2 h (line c ) . RNAs were hybridized to a "P-labeled UCP probe. Poly(A)+recovery of RNA hybridized to nitrocellulose was measured by hybridization to radioactive poly(T) probe. obese mice is also in agreement with previous studies where an almost normal physiological response to cold exposure or sympathomimeticdrugs wasmeasured (36, 38-42). Thus, although the thermogenic capacity of brown adipose tissue may be reduced (15, 16) or not (39) inobese mice maintained a t room temperature, the tissueis capable of responding to padrenoreceptor stimulation. Hencea low activity of the sympathetic nerves supplying the tissueof the obese mouse is the probable cause of defective brown fat regulatory thermogenesis in this animal. UCP mRNA in Obese falfa Rats-Several investigators have emphasized the low thermogenic capacity of brown adipose tissue in obese Zucker rats (15, 16). Radioimmunoassay measurement of UCP indicated that the proportion of UCP is decreased in mitochondria from adult or6-week-old fa/fa rats but not in their pups (37,43,44). Conversely, Bazin et al. (45) assayed a lower GDP binding to brown adipose tissue mito-

UCP mRNA Expression in Brown Adipose Tissue

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chondria of Zucker fa/fa pups aged 2-14 days. Moreover, a reduced proportion of LJCP was observed in electrophoresed mitochondrialproteins from these animals.:' Hereit is reported that. UCP mRNA is significantly lowered not only in 8- or 10-week-old obese rats but also in IO-day-old pups (Fig. 5 ) . Thus. UCP biosynthesis is altered in obese fa/fa rats and thisalteration is concomitantwiththeonset of obesit,y. Nevertheless the regulation of UCP biosynthesis infa/fa rats appears complicated since we measured a normal level of UCP mRNA in 5-week-old obese rats compared with lean littermat.es of the same age.' In order to assay the reactivity of brown fat of obese rats to stimulation, IO-week-old fa/fa rats were either exposed to cold or dosed with BRL 26830A for 2 h. In these animals a slightlv subnormal but striking increase was detected (Fig. 6). Thus the reduced amount of UCP mRNA in obese fa/fa rats is not due to brown adipocytes themselves but to understimulation of' cells by sympathetic nerves. lJCP m K N A in Obese Rats with Lesionof the VMH AreaThe existence of a functional link between the ventromedial hvpothalamic area and the interscapular brown adipose tissue

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FIG.6. Effect of stimulation by cold exposure of &agonist drug injection on UCP mRNA in genetically obese fa/fa rats; Northern analysis. Animals were 10 weeks old. Lanes I , 3,and 5 , RNA from controlleananimalsmaintained a t room temperature (lane 1 ) or dosed with BRL 26830A for 2 h(lane 3)or exposed a t 5 "C for2h (lane 5 ) . Lanes 2, 4, and 6, RNAfromobesefa/fa rats maintained a t room temperature (lane 2 ) or dosed with BRL 26830A for 2 h (lane 4 ) or exposed a t 5 "C for 2 h (lane 6). Poly(A)' recovery was assessed by hybridization to poly(T) and p41 cellular actin probes.

FIG. 5. UCP mRNA level in brown adipose tissue of young and obese adults from genetically obese fa/fa strain; Northern analysis. Left, poly(A)+ RNAfrom 10-day-old lean (1)and obese (2) rats. Right, poly(A)'RNAfrom8-week-oldlean (1) and obese (2) rats. The same data were obtained with 10-week-old lean and fa/fa rats. Arrouls indicate theposition of 1.9- and 1.5-kilobase pairs RNAs. Poly(A)' recovery was measured by hybridization to "'P-labeled poly(T) and p41 cellular actin probes. ~~

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R. Razin and D. Ricquier. data not shown. D. Ricquier, D. A. York, and R. Bazin, data not shown.

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has been demonstrated (46-48). Brown adiposetissue of obese VMH-lesioned rats is poorly thermogenic (49). Fig. 7 shows that the UCP mRNA level is reduced in VMH-lesioned rats but canbe increased after acuteexposure to cold. Such results agree with previous assays of UCP level and calorimetric measurement of the brown adipose tissue in these animals maintained a t room temperature or exposed to the cold (4951). Thus, studies on obese VMH rats lead to the conclusion previously obtained with other obese animals examined here, i.e. obese animals are able to activate UCP synthesis when brownadipocytes are correctly stimulated. Norepinephrine turnover in brown adipose tissue of obese animals tends tobe reduced but can be increased during cold exposure as in lean animals (15, 16, 52-54). Data presented here on UCP mRNA level in ob/ob mice, fa/fa rats, and VMH-lesioned rats provide further evidence that UCP regulation is essentially controlled by norepinephrine released in close proximity to thebrown adipocytes and that the primary lesion in obese animals is probably in the central nervous system which is responsible for chronic low sympathetic nerve activity in the tissue. Quantitation of the Relative Number of UCP Nascent Chains during Activation or Inhibition of Thermogenesis in Brown Adipose Tissue-In order to clarify the regulation of UCP expression, the rate of transcription initiation of Ucp gene was measured using isolated nuclei transcribed i n uitro. Nascent Ucp gene transcripts were identified in brown adipose

UCP m R N A Expression in Brown Adipose Tissue

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FIG. 9. Ucp gene transcription in isolated nuclei of brown adipose tissue from obese fa/fa rats; effect of BRL 26830A injection. Animals were 10 weeks old; RNA chains were elongated using the runoff transcription procedure. Labeled RNAs were hybridized to linearized UCP plasmid. Lean (Fa/fa) animals were either maintained at room temperature ( R T )or exposed at 5 "C for 15 min or dosed with BRL 26830A for 30 min. Obese (fa/fa) animals were maintained at room temperature or dosed with the &agonist compound. No experiment wasperformedwith obese rats exposed at 5 "C. Similar data were obtained when labeled t,ranscripts were hybridized to DNA immobilized on membrane after agarose electrophoresis and transfer.

lean RT lean 5°C

VMH RT VMH 5°C FIG. 7. UCP mRNA level in brown adipose tissue of rats with obesity-inducing VMH lesions. Upper part, Northern analysis. Lane 1, RNA from sham-operated animal maintained a t room temperature. Lane 2, RNA from VMH-lesioned rat a t room temperature. Lower part,dot-blot hybridization. 1 or 2 pg of total RNA were dotted onnitrocellulose and hybridized to UCPprobe. Sham-operated lean animals and VMH-lesioned rats were a t room temperature ( R T ) or exposed a t 5"Cfor 5 h. Poly(A)+ recoverywas measured by hybridization to poly(T) and p41 cellular actin probes.

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QI FIG. 8. Ucp gene transcription in isolated nuclei of rat brown adipose tissue; effect of stimulation by cold exposure or BRL 26830A injection. Nascent RNA chains from nuclei isolated from brown adipose tissuewere labeled by in vitro elongation and then hybridized with an excess amount of UCP, cellular actin, or pBR DNA. Left, dot analysis. Labeled RNA transcripts from brown fat nuclei (a-h) or from liver nuclei ( i ) were hybridized with linearized pUCP 36 plasmid. a, d, and g, animals maintained a t room temperature; b and e, animals exposed a t 5 "C for 15 min; c, rats dosed with BRL 26830A for 30 min; f and h, rats exposed a t 5 "C for 1 and 24 h, respectively. Right, Southern analysis. Labeled RNA transcripts from isolated brown fat nuclei were hybridized to large amounts of linearized p41 actin and pBR327 plasmids and UCP 36 insert immobilized on membrane after agarose electrophoresis and transfer; a, nuclei from control animals; b, nuclei from rats in cold for 15 min; c, nuclei from animals 30 min after BRL 2683019 injection.

tissue nuclei but not in liver nuclei (Fig. 8). No hybridization to UCP cDNAwas observed with RNA that was elongated in the presence of 1 pg/ml a-aminitin (data not shown). In the presence of this inhibitor, incorporationof radioactive [N-'~P] UTP into RNA transcripts was reduced by 74%. These data indicate that Ucp transcription is specific for brown adipose tissue and is dependent on RNA polymerase I1 activity. Fig. 8 shows that the concentration of nascent Ucp transcripts was strikingly increased when animals were exposed to cold for 15 min. The time course of induction of transcription reveals a strong rapid activation followed by a continuous but decelerated activation in tissue stimulated for a longer time. Thus, although these data do not rule out a possible posttranscriptional regulation of UCP mRNA. they demonstrate that Ucp gene is controlled at the transcriptionallevel. Interestingly BRL 26830A injection to rats is able to mimic the rapidincreaseinnascent Ucp transcript,s (Figs. 8 and 9). Moreover, the relative rate of Ucp transcription was reduced in obese fa/fa rats (Fig. 9) but was significantly increased when these animalswere dosed with the BRL26830A agonist (Fig. 9). All these data are in excellent agreement with data from Northern experiments and favor the existence of transcriptional regulation of Ucp. Increased or decreased UCP mRNA level in thermogenic or weakly thermogenic brown fat can be explained by the altered rateof Ucp gene transcription. Activation or inhibition of this process is controlled by 8adrenoreceptors of brown adipocytes. Reduced transcription of Tlcp gene in obese rats is not due to adefective gene nor to an altered cellular event but to limited nervous stimulation of the tissue. CONCLUSION

The availability of a cDNA clone containing a sequence complementary to UCP mRNA (21) facilitated the assay of changes in UCPmRNAunderdifferent physiological and pathological circumstances. Inductionof UCP mRNAis rapid following stimulation of the brown adipose tissue by exposure of animals to the cold. This induction is mediated by activation of the P-adrenoreceptors of plasma membrane of brown adipocytes. Moreover, in several types of obesity where sym-

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UCP mRNA Expression i n BrownAdipose Tissue

pathetic activity is known to be defective, the UCP mRNA either is reduced and/or can be increased during stimulation of the tissue by cold exposure or @-agonistdrug administration. Studies with isolated nuclei indicate that Ucp gene is regulated at the level of transcription. Nevertheless several questions remained unanswered. 1) Does norepinephrine/@ receptor interaction only induce UCP gene transcription or is a post-transcriptionalprocess also activated? 2) What are the intracellular factors which activate UCP mRNA synthesis? 3) What are the genomic sequences important in the regulation of this gene? These questions could be solved by in vitro experiments with isolated or cultured brown adipocytes, identification ofUcp promotor, and introduction of the cloned Ucp gene into different types of cells. The answers to these questions could help to understand the regulation of expression of a unique inducible mitochondrial protein capable of uncoupling substrate oxidation from ADP phosphorylation.

21. 22. 23. 24. 25. 26. 27. 28. 29.

Acknowledgments-We wish to thankChristine Blanchard, Eliane Hentz, and Marie-France Kinebayan for excellent technical assistance, Professor Jean Thibaultfor help in isolation of poly(A)+ RNA, Dr. Margaret Buckingham for the gift of cellular actin plasmid, Dr. Axel Kahn for the gift of aldolase B plasmid, Professor Antony Means for the gift of calmodulin plasmid, Dr. Mary Weiss and Dr. JeanLouis Nahon for helpful discussion concerning the runoff transcription procedure, Professor LeslieKozak for communication of an unpublished paper (Ref. 24), Sonia Cunningham for careful reading of the manuscript, and Martine Fernandez for typing.

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1. Nicholls, D. G., and Locke, R. (1984) Physiol. Reu. 64, 1-64 2. Nedergaard, J., and Cannon, B. (1984) in Bioenergetics (Ernster, L., ed) pp. 291-314, Elsevier Scientific Publishing Co., Amsterdam 3. Girardier, L. (1983) in Mammalinn Thermogenesis (Girardier, L., and Stock, M., eds), pp. 50-98, Chapman and Hall, London 4. Klingenberg, M. (1984) Biochem. SOC. Trans.12, 390-393 5. Ricquier, D., and Bouillaud, F. (1986) in Brown Adipose Tissue (Trayhurn, P., and Nicholls, D. G., eds) pp. 86-104, Edward Arnold (Publishers) Ltd., London 6. Ricquier, D., Thibault, J., Bouillaud, F., and Kuster, Y. (1983) J. Bwl. Chem. 258,6675-6677 7. Freeman, K.B., Chien, S.-M., Litchfield, D., and Patel, H. V. (1983) FEBS Lett. 158,325-330 8. Bouillaud, F., Ricquier, D., Mory, G., and Thibault, J. (1984) J. Bwl. Chem. 259,11583-11586 9. Freeman, K. B., and Patel, H. V. (1984) Can. J. Bwchem. Cell Biol. 6 2 , 479-485 10. Arnold, J., Patel, H.V., Ridley, R. G., and Freeman, K. B. (1985) Bwsci. Rep. 5 , 57-62 11. Freeman, K. B., Meyrick, K., Patel, H. V., and Ridley, R. (1985) Can. J. Biochem. Cell Biol. 6 3 , 988-991 12. Bouillaud, F., Weissenbach, J., and Ricquier, D. (1986) J. Biol. Chem. 2 6 1 , 1487-1491 13. Ricquier, D., and Mory, G . (1984) Clin. Endocrinol. Metab. 13, 501-519 14. Rothwell, N., and Stock, M. J. (1986) in Brown Adipose Tissue (Trayhurn, P., and Nicholls, D. G., eds) Edward Arnold (Publishers) Ltd., London, in press 15. Himms-Hagen, J. (1983) Nutr. Reu. 41,261-267 16. Trayhurn, P., and James, W. P. T. (1983) in Mammalian Thermogenesis (Girardier, L., and Stock, M. J., eds) pp. 234-258, Chapman and Hall, London 17. Mory, G., Ricquier, D., Nkhad, M., and Hinaon, P. (1982) Am. J. Physiol. 2 4 2 , C159-Cl65 18. Mory, G., Bouillaud, F., Combes-George, M., and Ricquier, D. (1984) FEBS Lett. 1 6 6 , 393-396 19. Young, P., Wilson, S., and Arch, J. R. S. (1984) Life Sci. 34, 1111-1117 20. Arch, J. R. S., Ainsworth, A. T., Ellis, R. D. M., Piercy, V.,

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