Molecular studies of the uncoupling protein. - The FASEB Journal

Molecular

studies

D. RICQUIER,’

of the uncoupling

L. CASTEILLA,

Centre de Recherche sur I’Endocrinologie

AND

F. BOUILLAUD

Mol#{233}culaire et le D#{233}velo/,pement,CNRS-UPR

ABSTRACT The uncoupling protein (UCP) is a protonl anion transporter found in the inner mitochondrial membrane of brown adipocyte. Although UCP has not been detected in mitochondria from any other tissue, it shares structin-al and catalytic properties with several other mitochondrial carrier proteins. Although UCP was discovered only recently it is one of the most extensively studied mitochondrial carrier proteins. Many tools useful in research on UCP have been developed such as antibodies and cDNAs corresponding to UCP of several animal species. More recently, the mouse, rat, and human genes encoding for UCP have been isolated and sequenced. The availability of these various tools has led to several significant observations. UCP gene expression is strongly controlled at the level of transcription by signals that are activated after the stimulation of brown adipocytes by norepinephrine. The comparison of UCP gene with the genes encoding the adenine nucleotide translocator revealed the existence of structural and evolutionary homologies. Moreover, in humans the UCP gene and one form of adenine nucleotide translocator gene are located on the same chromosome. Recently, the expression of functional UCP in various heterologous systems was achieved (Xenopus oocytes, CHO cells, yeasts). These data will facilitate studies of the structure/function relationship in UCP (identification of residues involved in W transport, Cl transport, nucleotide binding, mitochondrial targeting. . .). Another aspect of the present research on UCP is the understanding of mechanisms that control the UCP gene and the differentiated commitment of adipose precursor cells to thermogenic brown adipocytes. The multifaceted aspects of research on UCP make this protein interesting in areas of research as different as studies of ion translocating mechanisms, cellular specificity of gene transcription, control of gene expression by neuromediators, adipocyte differentiation, and the pharmacological treatment of obesity. Ricquier, D.; Casteilla, L.; Bouillaud, F. Molecular studies of the uncoupling protein. FASEBJ. 5:

2237-2242; Key Words:

1991. mitochondria

.

transport

.

adipocyte

thermogenesis

DISCOVERY OF THE UNCOUPLING protein (UCP)2 revealed one type of mechanism developed by mammals to resist cold and regulate body temperature. UCP, which is uniquely present in brown adipocytes, is very active at birth in most mammals (including human infants), during cold exposure in rodents, and during arousal in hibernators. The main part of what is known about UCP comes from the interactive studies by physiologists and bioenergeticists. It appeared that research on UCP was of interest for several reasons, including 1) the mechanism of assembly into the mitochondrial membrane of a protein that has no NH2-terminal targeting sequence, 2) the functional Organization of a proton translocator, 3) the regulation of ion transporters by nucleotides, 4) the cell specificity of gene expression and the regula‘ThE

0892-6638191/0005-22371$O1.50.

©

FASEB

protein 1511, 92190 Meudon, France

tion of a gene by neuromediators, and 5) the development of thermogenic drugs active against obesity via their effects on UCP and brown adipoctye differentiation. Between 1961 and 1965 it had been demonstrated that one distinct form of adipose tissue, the brown adipose tissue (BAT), was in fact a thermogenic organ in rodents and lagomorphs (see ref 1). Subsequently it was demonstrated that heat production by brown adipocytes resulted from a controlled uncoupling of oxidative phosphorylation. In a series of elegant experiments, Nicholls (1) demonstrated that uncoupled BAT mitochondria were characterized by a unique proton conductance pathway operating in the inner mitochondrial membrane. Activation of this pathway dissipates the proton electrochemical gradient generated in mitochondna by respiration short-circuiting the ADP phosphorylation system, resulting in energy dissipation as heat. The proton transport pathway was identified as a 32-kDa protein uniquely present in BAT mitochondria and generally named the uncoupling protein or UCP. Previously, it had been reported that a 32-kDa protein of the membrane of BAT mitochondria was strongly induced in rats exposed to cold. It was also demonstrated that UCP activity was inhibited by purine nucleotides (GDP, GTP, ADP, or ATP) and that UCP synthesis as well as brown fat development were stimulated by norepinephrine (see reviews in refs 1-4). In this brief review we will discuss the strategies used to study UCP and summarize knowledge on physiological, biochemical, and genetic aspects of UCP. Detailed data on carrier mechanisms will not be discussed here (see refs 5 and 6).

TOOLS

TO

STUDY

THE

UNCOUPLING

PROTEIN

For a long time the assay of UCP was based on the measurement of GDP binding to isolated brown fat mitochondria. The protein was also characterized by photoaffinity labeling with radioactive azidonucleotides (7-9; see Fig. 1). The catalytic activity of UCP was assayed through measurement of the proton conductance of the mitochondrial membrane of brown fat cells (2). The purification of UCP was developed in Klingenberg’s laboratory (10). This purification allowed the development of antibodies against rodent and human UCP (Fig. 1). Then Bouillaud et al. (11) reported the isolation of a full-length rat UCP cDNA. Others have also isolated partial mouse UCP cDNA (12) and complete rat UCP cDNA (13). More recently, bovine (14, 15) and rabbit UCP cDNA (16) were also characterized. Mouse (17), rat (18), ovine (14), and human (19, 20) UCP genes were recently isolated and entirely or partially sequenced. Thus, a great variety of antibodies, cDNA, and genomic probes corresponding

‘To whom correspondence should be addressed, at: Centre de Recherche sur l’Endocrinologie Mol#{233}culaire et le D#{233}veloppement, CNRS, 9 rue Jules Hetzel, F-92190 Meudon, France. 2Abbreviations: ANT, adenine nucleotide translocator; BAT, brown adipose tissue; CHO cells, Chinese hamster ovary cells; UCP, uncoupling protein.

2237

A: Photoaffinity

Immunodetection

B:

labelling

a

b

C

or 20p

12345

_actiri

_i.gkb _pBR

_1.6kb

ucP

C: UCP

mRNA

D: Run-on

analysis

transcription

Figure 1. Identification of UCP and characteristics of UCP synthesis. A) Photoaffinity labeling with [‘2P]8-azido ATP of mitochondria isolated from human brown adipose tissue. UCP is identified as a 32-kDa protein, distinct from the ADP/ATP carrier (30-kDa molecular weight). Data from ref 8 with permission. B) Western blot analysis of human (lanes a and b) and rat (lane c) UCP using antibodies raised against rat UCP. 50 and 25 sg protein were used in lanes a and b and 2 g in lane c. Unpublished data from Garruti and Ricquier. C) Northern blot analysis of UCP mRNAs in BAT of rats exposed to cold for 0 h (lane 1), 1 h (lane 2), 5 h (lane 3), and 24 h (lane 4). No UCP mRNA could be detected in liver RNA (lane 5). RNAs were probed with a rat UCP cDNA. Data from ref 24 with permission. D) Run-on transcription assay of UCP gene in nuclei isolated from brown adipose tissue of control rat (lane a), rat exposed at 5#{176}C for 15 mm (lane b) or acutely treated with -adrenoceptor agonist (lane c). Data from ref 24, with permission. to UCP of various species are presently available. As was pointed out by Kramer and Palmieri (21) it is surprising to have so much data on UCP, a recently discovered mitochondrial transporter. With the exception of the adenine nucleotide translocator (ANT), a limited number of tools corresponding to other mitochondrial carriers have been characterized.

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June 1991

MAIN

CHARACTERISTICS

UNCOUPLING

UCP is uniquely

OF THE

PROTEIN present

in brown

adipocytes

Using antibodies or cDNA probes (Fig. 1), several laboratories were unable to detect UCP or its mRNA in cells other than brown adipocytes (4, 11, 12). These data are in agree-

The FASEB Journal

RICQUIER

ET AL.

ment with previous studies showing that compared with mitochondria from other tissues, brown fat mitochondria were characterized by a GDP-sensitive uncoupled respiration and a GDP-sensitive high proton conductance (2). UCP is an excellent marker of brown adipocytes and also confers on such cells their unique capacity to dissipate a large quantity of oxidation energy as heat. It is now accepted that any cell containing UCP is a brown adipocyte.

and the brown fat is replaced by adipose tissue phenotype of the white adipose tissue. Apparently mammals the mechanisms that control UCP gene are rapidly altered at birth. However, observations adult patients with pheochromocytoma (3) or of treated with certain /3-adrenoceptor agonists (32, indicate that the UCP gene remains responsive to stimulation (Fig. 2).

UCP synthesis

The uncoupling protein multigene family

and

T3,

and

is inducible, transcriptionally

controlled by norepinephrine regulated

UCP appears before birth in the BAT of most mammals (4, 22). The mechanisms that control brown adipocyte differentiation and UCP gene expression are poorly understood. This was due mainly to the failure to isolate cell lines derived from the brown adipocytes that express UCP. However, Rehnmark et al. (23) have recently reported the full differentiation of brown adipocytes expressing UCP in primary cultures derived from fibroblastic precursor cells isolated from the interscapular brown fat of young mice. In vivo, UCP level can be modulated by various physiological stimuli. The most striking example is that of exposure of rats to cold which triggers a rapid and marked increase in UCP mRNA and UCP levels (11, 12, 24; Fig. 1). In most rodents, an increase in nonshivering thermogenesis capacity is accompanied by the parallel increase in UCP synthesis. Birth in rodents is another example of a physiological stimulus that stimulates UCP synthesis (24, 25). Moreover, it has also been shown that nutritional changes such as refeeding after starvation increase the UCP level (26, 27) and activate UCP mRNA synthesis (28). These data agree with the proposal that brown fat could buffer a part of energy corresponding to food intake and could be a regulator of body weight (1, 26, 27). The rapid stimulation of UCP synthesis in animals exposed to cold suggested a transcriptional control of the UCP gene and also raised a question as to the nature of the signals involved in this activation. Recent studies have provided at least partial answers to these questions. Run-on transcription experiments with nuclei isolated from BAT of rats demonstrated that UCP gene was essentially regulated at the transcriptional level because exposure of rats at 4#{176}C for 15 mm induced a 10-fold increase in the initiation rate of UCP gene transcription (24) (Fig. 1). It was also shown that acute treatment with fl-adrenoceptor agonist (24) or norepinephrine (29) induced a similar increase in UCP gene transcription. Recently, Rehnmark et al. (23) demonstrated that norepinephrine activates the transcription of UCP gene in brown adipocytes differentiated in culture. These data confirmed previous experiments with rats dosed with norepinephrine, and observations made of rats and humans bearing the norepinephrine-secreting tumors pheochromocytoma, indicating that this catecholamine was the main physiological activator of UCP synthesis and BAT development (3). However, Bianco et al. (29) have demonstrated that a high level ofT3 produced through deiodination ofT4 by 5’-deiodinase in brown adipocytes was necessary to obtain the optimal effect of norepinephrine on UCP gene transcription. Conversely, inhibition of T4 5’-deiodinase of brown adipose tissue by iopanoic acid impaired the coldinduced increase in UCP and UCP mRNA (30, 31). Rats have active BAT at all stages of life. The situation in large animals such as ovines, bovines, dogs, and probably humans is different. At birth, almost all fat deposits in lambs and calves contain a high level of UCP mRNA and UCP (9, 14). Curiously, within the very first days of life the UCP mRNA disappears (14), then the UCP level decreases (9),

UNCOUPLING

PROTEIN

is a member

having the in large expression made of adult dogs 33) clearly adrenergic

of a

One of the most striking conclusions from the sequencing of purified UCP (34) or cloned UCP cDNAs (13, 35) was that UCP is strongly homologous to several other ubiquitous, mitochondrial carriers. This point was recently reviewed (5, 36) and will not be detailed here. UCP was the second mitochondrial transporter to be sequenced after the ANT (36). The third sequenced mitochondrial carrier was the phosphate carrier (36, 37). It was concluded that these three transporters derive by triplication from a common ancestor. It is also known that the oxoglutarate carrier is a member of the same protein family (21, 37). Moreover, another protein referred to as hML-7, whose function is unknown, has been cloned and its cDNA sequenced (38). This protein is a major antigen in autoimmune Grave’s disease and is obviously related to other sequenced mitochondrial carriers, which suggests that it is also involved in transport of anions through the inner mitochondrial membrane (38). It is known that most mitochondria possess 12-15 different membranous carriers. The primary sequence of only fout carriers has been determined. Because these four transporters are obviously related, one may speculate that several other mitochondrial transporters belong to the same gene family.

NB ADULT

NB ADULT

4

-#{247}

-

-

-t

!

,,

‘-“4’

ucP

4i

UCPmRNA

Figure 2. Lack of UCP and UCP mRNA in perirenal adipose of control adult dog and reinduction after 2 wk treatment with the thermogenic drug LY79730. NB, newborn dog; -, adult dog treated with placebo; +, adult dog receiving the thermogenic drug. UCP was immunodetected in 50 cg of mitochondrial protein isolated from perirenal adipose tissue. UCP mRNA was analyzed by Northern blot procedure using hybridization of 20 ig RNA to rat UCP cDNA. 0. Champigriy, B. R. Holloway, and D. Ricquier, unpublished results.

2239

0

10

20

40

Cp

Ru Mouse Human

ANT

MaSzel,2

140

300

175

240

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iD

02*

*]f

)

J(D3*

Human I

Neurospora

275

)

*4

(-r)(;#{149}-(--;#{149} 0*1 0*3

If Figure 3. Alignment of uncoupling protein (UCP) and adenine nucleotide transi#{243}cator (ANT) genes. Each gene is split into 3, 4, or 6 exons (numbered 1 to 6, from the 5’ to the 3’ end). The thin arrows indicate the position of amino acids corresponding to the extremities of the exons. The two thick arrows delineate the three repeated domains I, II, and III. Open square boxes and asterisks indicate the position of two peptidic motifs that are strongly conserved in the different proteins (0, P-D-VK-R motif; ‘, G-FKG motif).

Figure 4 is an attempt to represent the hypothetical 100 amino acid-long ancestor of the different mitochondrial carriers. We propose that this ancestral protein was made of several subdomains. The first domain is made of a glycinealanine rich region attached to the P-D-V(K/R)-R motif. Amino acids contained in this domain could form an a-helix. The second domain contains a tyrosine residue and the EG-aromatic residue (K/R)G motif. This region, which is homologous to an ADP binding site in ANT (41), could be involved in nucleotide binding. The rest of the domain does not present a particular organization. However, one cannot rule out the possibility that the ancestral protein was shorter than 100 amino acids. It is at present difficult to elucidate the different steps in the evolution of mitochondrial carriers. This is particularly so because it is obvious that the different carriers have not evolved at the same speed. In particular, we have calculated that UCP has evolved 7- or 8-fold more rapidly than ANT (collaboration with G. Lecointre, University of Orsay, France).

CONCLUDING

Comparison

of UCP

and

ANT

genes

In the last 2 years, the genes encoding rodent UCP (17, 18), human UCP (19, 20), and human ANT (39, 40) have been cloned and sequenced. There is at present no report on the isolation of the mitochondrial phosphate carrier gene. The situation is simple for UCP because it is coded for by a single gene (11, 12, 20). Conversely, the situation is much more complicated for mammalian ANT, which has several isoforms. In mammals the expression of these isoforms is the consequence of the activity of at least three different ANT genes (ANTi, ANT2, and ANT3). The heart-skeletal muscle ANTi gene in humans has been assigned to chromosome 4 using both flow-sorted chromosomes and segregating humanmouse hybrid cells (39). The human UCP gene has also been assigned to the long arm of chromosome 4 in q31 (20). In Fig. 3 we tried to align UCP and ANT genes. The UCP gene is split into six exons and each repeated domain of 100 amino acids (I, II, and III) is encoded by two exons separated by one intron. The triplicated structure of the maize ANT gene, which is made of three exons, is also obvious. It is more difficult to correlate the tripartite structure of the mammalian ANT with a tripartite organization of corresponding genes. However, several limits of exon present in UCP genes were found in ANT genes from maize, human, and Neurospora cnzssa (positions 40 and 200 in Fig. 3). The different exons corresponding to the two transporters share highly conserved peptide domains indicated by an open square and a star in Fig. 3.

Positions of introns In mitochondrial carrien:

ANT:

REMARKS

AND

The main characteristics of UCP are 1) its unique expression in one particular type of cell, 2) its regulated proton (and anion) translocating activity, and 3) its evolutionary relationship with other mitochondrial carriers. These features make UCP a remarkable protein with which to study several important scientific questions such as the cell specificity of gene expression, the different steps of differentiation of a particular type of adipocyte, and the function organization of an ion transporter of the mitochondrial membrane. Regulation differentiation

of UCP

gene

expression

brown

adipocyte

(fl-rn) Us, Zm UCP:(I-II)et(ll-fl1)

(1J Nc,Hs

ANT:

uCP:(fl)et(m

UOIls UCP: (1)

Gly-Ala rich

and

Several groups are presently trying to identify genomic regulatory elements involved in the regulation of UCP gene. Their approach is based on transfection of cells with constructions of the 5’ region of the UCP gene fused to chloramphenicol acetyltransferase. The main objective of research on UCP gene regulation is to understand why UCP synthesis is unique to mature brown adipocytes. In fact this question is equivalent to the question of what makes pluripotent precursor cells committed to brown adipocytes. The correct answer will depend on identification of the mechanisms responsible for such a commitment. Such research may lead to a better understanding of determinants of obesity, and offers insight into novel approaches to the pharmacological treatment of obesity. In rodents, activation of BAT thermogenesis promotes a reduction in body weight gain (2, 26, 27), and in large mammals BAT is apparently converted during

11, Hypothetical 100 amIno-acid long ancestor

PERSPECTIVES

________

P-D-V+-R Y

---lIGI

ANT:

(I) Zm

I

Figure 4. Proposed structure of the hypothetical protein ancestor of mitochondrial carriers. This protein, made of 100 or fewer amino acids, contained regions conserved in mitochondrial transporters. The hatched box corresponds to a region deleted in the phosphate carrier that is highly variable in mitochondrial carriers. Arrows indicate the positions corresponding to introns in mitochondrial carriers (Nc, Neurospora crassa; Hs, humans; Zm, Zea mays). Roman numerals identify repeated domains (I, II, and III) in mitochondrial transporters.

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RICQUIER

ET AL.

aging to white adipose tissue whose function is to store energy. Several new /3-adrenoceptor agonists, often classified as new thermogenic drugs, have a slimming effect in animals (33, 42, 43). These drugs activate UCP gene transcrption (24) and UCP synthesis in rodents (24, 43). In adult dogs they induce the development of BAT (33; and 0. Champigny, D. Ricquier, and B. R. Holloway, unpublished results). It remains to be demonstrated if such molecules activate quiescent precursor cells or stimulate the expression of UCP by established adipocytes. The

functional

organization

of UCP

UCP, ANT, and the mitochondrial phosphate carrier not only have homologous primary structure but share several functional characteristics. UCP and ANT do not have an NH2-terminal cleavable targeting sequence; moreover, both proteins bind di- and triphosphate purine nucleotides. UCP and the mitochondrial phosphate carrier are H/OH transporters. Thus any progress in understanding the functional organization of UCP will certainly be of benefit in understanding the transport mechanism and regulation of other mitochondrial carriers. It has been demonstrated that UCP has at least two internal mitochondrial targeting signals, one located within the NH2-terminal third of the molecule and the other downstream of this position (44). The same group has demonstrated that the ability of UCP to insert into the inner membrane was abrogated when the molecule was fused behind a matrix-targeting signal (45). A powerful strategy to identify catalytic or regulatory domains in UCP is the expression of normal or mutated UCP in heterologous systems. Such an approach is greatly facilitated by the fact that UCP exists solely in brown adipose cells. Recently, the expression of normal UCP was obtained in Xenopus oocytes, CR0 cells, and yeast. In the first system, expressed UCP was inserted into mitochondrial membranes but its uncoupling activity could not be measured (46). Stable CHO cells lines that express functional UCP were isolated (47). In mitochondria isolated from these cells, UCP was able to uncouple respiration and lower the membrane potential; in addition, the uncoupling activity of UCP was inhibited by GDP. In yeasts expressing UCP (48), the binding of GDP to isolated mitochondria could be demonstrated. These expression systems could be used to check the activation of UCP by free fatty acids as proposed by Rial and Nicholls (49). Further studies of mutated UCP will also help reveal the intramembrane organization of UCP and identify residues involved in nucleotide binding and proton or anion transport. Finally, one may hope that research on UCP will contribute to the biochemistry of other mitochondrial transporters. Our work is supported by CNRS, INSERM, DRET, and Association de Recherche sur le Cancer. We express our gratitude to Drs. G. Brandolin, M. Douglas, K. Garlid, R. Kramer, D. L. Kohn, F. Palmieri, P. Pedersen, E. Shrago, D. Wallace, and J. E. Walker for communication of reprints and preprints. We are indebted to Dr. B. Holloway and Mrs. R. Mayers for expert reading of the manuscript and to Patricia M#{233}ralli for secretarial assistance. REFERENCES 1. Nicholls, D. G., and Locke, R. M. (1984) Thermogenic mechanisms in brown fat. Physiol. Rev. 64, 1-64 2. Himms-Hagen, J. (1990) Brown adipose tissue thermogenesis: interdisciplinary studies. FASEBJ. 4, 2890-2898 3. Ricquier, D., and Mory, G. (1984) Factors affecting brown adi-

UNCOUPLING

PROTEIN

pose tissue activity in animals and man. Gun. Endocrinol. Metab. 13, 502-519 4. Nedergaard, J., Connolly, E., and Cannon, B. (1986) Brown adipose tissue in the mammalian neonate. In Brown Adipose Tissue (Trayhurn, P., and Nicholls, D. G., eds) pp. 152-213, Arnold, London 5. Klingenberg, M. (1990) Mechanism and evolution of the uncoupling protein of brown adipose tissue. TIBS 15, 108-112 6. Garlid, K. D. (1990) New insights into mechanism of anion uniport through the uncoupling protein of brown adipose tissue mitochondria. Biochim. Biophys. Ada 1018, 151-154 7. Heaton, G. M., Wagenvoord, R. J., Kemp, A., and Nicholls, D. G. (1978) Brown adipose tissue mitochondria: photoaffinity labelling of the regulatory site of energy dissipation. Eur. j Biochem. 82, 515-521 8. Bouillaud, F., Combes-George, M., and Ricquier, D. (1983) Mitochondria of adult human brown adipose tissue contain a 32000Mr uncoupling protein. Biosci. Rep. 3, 775-780 9. Casteilla, L., Forest, C., Ricquier, D., Robelin, J., Lombet, A., and Ailhaud, G. (1987) Characterization of the mitochondrial uncoupling protein in bovine foetus and newborn calf. Disappearance in lamb during aging. Am.]. Physiol. 252, E627-E636 10. Lin, C. S., and Klingenberg, M. (1980) Isolation of the uncoupling protein from brown adipose tissue mitochondria. FEBSLetI. 113, 299-303 11. Bouillaud, F., Ricquier, D., Thibault, J., and Weissenbach, J. (1985) Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein. Proc. Nail. Acad. Sd. USA 82, 445-448 12. Jacobsson, A., Stadler, U., Glotzer, M., and Kozak, L. (1985) Mitochondrial uncoupling protein from mouse brown fat. Molecular cloning, genetic mapping and mRNA expression. J. BioL Chem. 260, 16250-16254 13. Ridley, R. G., Patel, H. V., Gerber, G. E., Morten, R. C., and Freeman, K. B. (1986) Complete nucleotide sequence and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of mitochondrial targeting pre-sequence. Nucleic Acids Res. 14, 4025-4035 14. Casteilla, L., Champigny, 0., Bouillaud, E, Robelin, J., and Ricquier, D. (1989) Sequential changes in the expression of mitochondrial protein RNA during the development of brown adipose tissue in bovine and ovine species. Bioc/jem. j 257, 665-671 15. Casteilla, L., Bouillaud, F., Forest, C., and Ricquier, D. (1989) Nucleotide sequence of a cDNA encoding bovine brown fat uncoupling protein. Homology with ADP binding site of ADP/ATP carrier. Nucleic Acids Res. 17, 2131 16. Balogh, A. G., Ridley, R. G., Patel, H. V., and Freeman, K. B. (1989) Rabbit adipose tissue uncoupling protein mRNA: use of only one of the two polyadenylation signals in its processing. Biochem. Biophys. Rev. Commun. 161, 156-161 17. Kozak, C. P., Britton, J. H., Kozak, U. C., and Wells, J. M. (1988) The mitochondrial uncoupling protein gene. Correlation of exon structures to transmembrane domains. J. BioL Chem. 263, 1274-1277 18. Bouillaud, F., Raimbault, S., and Ricquier, D. (1988) The gene for rat uncoupling protein: complete sequence, structure of primary transcript and evolutionary relationship between exons. Biochem. Biophys. Res. Commun. 157, 783-792 19. Bouillaud, F., Villaroya, F., Hentz, E., Raimbault, S., Cassard, A. M., and Ricquier, D. (1988) Detection of brown adipose tissue uncoupling protein mRNA in adult patients by a human genomic probe. Clin. Sci. 75, 21-27 20. Cassard, A. M., Bouillaud, F, Mattei, M. G., Hentz, E., Raimbault, S., Thomas, M., and Ricquier, D. (1990) Human uncoupling protein gene: structure, comparison with rat gene and assignment to the long arm of the chromosome 4. J. Cell Biochem. 43, 255-264 21. Kramer, R., and Palmieri, F. (1989) Molecular aspects of isolated and reconstituted carrier proteins from animal mitochondna. Biochem. Biophys. Ada 974, 1-23 22. Houstek, J., Kopecky, J., Baudysova, M., Janikova, D., Pavelka,

2241

S., and Klement, P. (1990) Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture. Biochim, Biophys. Acta 1018, 243-247 23. Rehnmark, S., N#{233}chad,M., Herron, D., Cannon, Nedergaard, J. (1990) a and $-Adrenergic induction pression of the uncoupling protein thermogenin

adipocytes differentiated 16464-16471

in

culture.

j

BioL

B., and of the exin brown

Chem.

265,

24. Ricquier, U, Bouillaud, F., Toumelin, P., Mory, G., Bazin, R., Arch, J., and P#{233}nicaud, L. (1986) Expression of uncoupling mRNA in thermogenic or wealdy thermogenic brown adipose tissue. j BioL Chem. 261, 13905-13910 25. Obregon, M. J., Jacobsson, A., Kirchgessner, T., Schotz, M. C., Cannon, B., and Nedergaard, J. (1989) Postnatal recruitment of brown adipose tissue is induced by the cold stress experienced by pups. Biochem. J. 259, 341-346 p. (1986) Brown adipose tissue and energy balance. In Brown Adipose Tissue (Trayhurn, P., and Nicholls, D. G., eds) pp. 299-338, Arnold, London 27. Rothwell, N. J., and Stock, M. J. (1986) Brown adipose tissue and diet induced thermogenesis. In Brown Adipose Tissue (Trayhum, P., and Nicholls, D. G., eds) pp. 269-298, Edward Arnold, London 26. Trayhurn,

28. Champigny, 0., and Ricquier, D. (1990) Effects of fasting and refeeding on the level of uncoupling protein mRNA in rat brown adipose tissue. Evidence for diet-induced and coldinduced thermogenesis. j Nuir. 120, 1730-1735

29. Bianco, A. C., Sheng, X., and Silva, E. (1988) Tniiodothyronine amplifies norepinephrine stimulation of uncoupling protein gene transcription by a mechanism not requiring protein synthesis. j Biol. Chem. 263, 18168-18175 30. Bianco, A. C., and Silva, J. E. (1987) Intracellular conversion of thyroxine to triiodothyronine is required for the optimal thenmogenic function of brown adipose tissue. j Clin. Invest. 79, 295-300 31. Reiter, R.J., Klaus, S., Ebbinghuas, C., Heldmaier, G., Redlin, U., Ricquier,

D., Vaughan,

M.

K., and

Steinlechner,

S. (1990)

Inhibition of 5’-deiodination of thyroxine suppresses the coldinduced increase in brown adipose tissue messenger ribonucleic acid for mitochondnial uncoupling protein without influencing lipoprotein lipase activity. Endocrinology 126, 2550-2554 32. Ashwell, M., Stnibling, D., Freeman, S., and Holloway, B. R. (1987) Immunological, histological and biochemical assessment of brown adipose tissue activity in neonatal, control and 3stimulant-treated adult dogs. Intern. j Obesity 11, 357-365 33. Holloway, B. R. (1989) Reactivation of brown adipose tissue. Proc. Nutr. Soc. 48, 225-230 34. Aquila,

H.,

Link,

T. A., and

Klingenberg,

M.

(1985)

The

un-

coupling protein from brown fat mitochondria is related to the mitochondnial ADP/ATP carrier. Analysis of sequence homologies and folding of the protein in the membrane. EMBO j 4, 2369-2376 35. Bouillaud, F, Weissenbach, J., and Ricquier, D. (1986) Complete cDNA derived amino acid sequence of rat brown adipose tissue uncoupling protein. j BioL Chem. 261, 1487-1490

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36. Aquila, H., Link, T., and Klingenberg, M. (1987) Solute carriers involved in energy transfer of mitochondnia form a homologous family. FEBS Lelt. 212, 1-9 37. Runswick, M. J., Powell, J. T., Nyren, P., and Walker, J. E. (1987) Sequence of the bovine mitochondrial phosphate carrier: structural

relationship

to ADP!ATP

translocase

and

the brown

fat mitochondnia uncoupling protein. EMBO]. 6, 1367-1373 38. Zarrilli, R., Oates, E. L., McBride, 0. W., Lerman, M. I., Chan, J. Y., Santisteban, P., Ursini, M. V., Notkins, A. L., and Kohn,

L. D. (1989)

Sequence

and

chromosomal

assignment

of

a novel cDNA identified by immunoscreening of a thyroid expression library: similarity to a family of mitochondnial solute carrier proteins. MoL Endocrinol. 3, 1498-1508 39. Li, K., Warner, C. K., Holge, J. A., Minoshima, S., Kudoh, J., Fukuyama, R., Maekawa, M., Shimizu, Y., Shimizu, N., and Wallace, D. G. (1989) A human muscle adenine nucleotide translocator gene has four exons, is located on chromosome-4, and is differentially expressed. j BioL Chem. 264, 13998-14004 40. Cozens, L. A., Runswick, M. J., and Walker, J. E. (1989) DNA sequences of two expressed nuclear genes for human mitochondna! ADP/ATP translocase. j Mol. BioL 206, 261-280 41. Dalbon, P., Brandolin, G., Boulay, F, Hoppe, J., and Vignais, P. V. (1988) Mapping of the nucleotide binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling

with 2-azido-[a-32P] adenosine diphosphate. Biochemistry 27, 5141-5149 42. Arch, J. R. S. (1989) The brown adipocyte /3-adrenoceptor. Proc. Nutt Soc. 48, 215-223 43. Muzzin, P., Revelli, J. P., Ricquier, D., Meier, M. K., Assimacoupoulos-Jeannet, F, and Giacobino, J. P. (1989) The novel thermogenic 3-adrenergic agonist Ro 16-8714 increases the interscapular brown-fat /3-receptor-adenylate cyclase and the uncoupling protein mRNA level in obese (fa/ja) Zucker rats.

Bioc/zem. j 261, 721-724 44. Liu, X., Bell, A. W., Freeman, K. B., and Shore, G. C. (1988) Topogenesis of mitochondnial inner membrane uncoupling protein. Rerouting transmembrane segments to the soluble matrix compartment. j Cell BioL 107, 503-509 45. Liu, X., Freeman, K. B., and Shore, G. C. (1990) An amino acid signal sequence abrogates the intrinsic membranetargeting information of mitochondnial uncoupling protein. j Biol. Chem. 265, 9-12 46. Klaus, S., Casteilla, L., Bouillaud, F., Raimbault, S., and Ricquier,

D. (1990)

coupling

protein

Expression

dna! membrane. Biochem. 47. Casteilla, L., Blonde!, 0.,

Bouillaud, tional ovary

of the brown

fat mitochondnial

in Xenopuc oocytes and import

F, and Ricquier,

Biophys. Res. Commun. Klaus, S., Raimbault,

167, 784-789 S., Moreau, F,

D. (1990) Stable expression

mitochondrial uncoupling protein cells. Proc. Nail. Acad. Sci. USA 87,

un-

into mitochon-

in Chinese

of funchamster

5124-5128 48. Pnieto, S., Bouillaud, F., Raimbault, S., Rial, E., Gimenez, G., and Ricquier, D. (1990) Expression of the brown fat mitochondrial uncoupling protein in S. cerevisaie. EBEC Rep. 6, 32 49. Rial, E., and Nicholls, D. G. (1987) The uncoupling protein from brown adipose tissue mitochondnia. Cell. Biol. Rev. 11, 75-104

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