International Journal of Obesity (1999) 23, Suppl 6, S19±S23 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo UCP1, UCP2 and UCP3: Are they true uncouplers of respiration?

F Bouillaud1*

1CEREMOD, CNRS, Meudon, France

The thermogenesis in brown adipose tissue (BAT) is due to the activity of a mitochondrial uncoupling (UCP1). This protein allows the protons pumped by the respiratory chain to re-enter the matrix without ATP synthesis. Therefore respiration is dramatically increased and produces only heat. The discovery of showing strong similarities with the UCP1 and expressed in other tissues raised the possibility that these participate in the proton leak observed in mitochondria, and therefore participate in the regulation of energy expenditure. The recombinant expression of UCP1, UCP2 and UCP3 in yeast allows the comparison of the coupling state of yeast mitochondria in the presence or absence of these proteins.

Keywords: mitochondria; proton leak; yeast; Saccharomyces cerevisiae

Introduction inhibit or mask naturally present uncoupling mechan- isms of physiological relevance. An example is BAT mitochondria, where the UCP1 is The ®rst hypothesis proposed with discovery of responsible for heat production. sequences highly similar to the brown adipose tissue (BAT) uncoupling protein (UCP1),1±4 was that these proteins had a similar activity. In this paper we will re-examine several results obtained with UCP1 that are relevant here, and will present the present status of The archetype of uncoupling the comparison between UCP2, UCP3 and UCP1. proteins: UCP1

Isolated BAT mitochondria proved to be largely Coupled mitochondria uncoupled, it took some time to be convinced that this uncoupling re¯ected a physiologically relevant thermogenic mechanism, the discovery of regulators Isolated mitochondria start to respire as soon as of this uncoupling was a key issue, for review see substrates and oxygen are available, and therefore Refs.5,6 Later on reconstitution of UCP17 and recom- the energy of substrates is lost. Addition of ADP binant expression8 showed that UCP1 is the explana- greatly accelerates respiration and an important part tion for the fatty acid (FA) induced and nucleotide of the energy liberated by the oxidation of substrates inhibited, uncoupling pathway of BAT mitochondria. is stored into ATP. This implies a coupling between Finally, it was observed that KO mice for the ICP1 oxidation and phosphorylation, and the controlled gene had a defective cold induced thermogenesis and, permeability to protons of the inner membrane is therefore, are cold intolerant.9 essential for this coupling. The ratio between the UCP1 activity results in a transport of protons respiratory rate in the presence or absence of ADP through the mitochondrial inner membrane. The (respiratory control ratio) indicates how mitochondria exact mechanism is still not completely understood. are coupled. More coupled mitochondria are consid- There is evidence from the literature that UCP1 is able ered as better accordingly biochemical procedures to transport many other solutes.10 Therefore if UCP1 were settled to increase the coupling state of mito- would have been just sequenced as an `orphan gene', chondrial preparations, but one can also examine the its role as a thermogenic protein would not have been possibility that some of the biochemical tricks used to easy to deduce on the basis of biochemical experi- improve the coupling state of mitochrondria, could ments. The physiologically relevant regulators of UCP1 are probably the FA that strongly activate the proton transport by UCP1 (for review, see Ref. 6 A *Correspondence: Dr F Bouillaud, CEREMOD, CRNS, 9 rue Jules Hetzel, 92190 Meudon, France. proton conductance dramatically increased by FA, and E-mail: [email protected] inhibited by GDP, indicates the presence of a UCPs as uncouplers F Bouillaud S20 functional UCP1.11 Therefore determination of speci- diminution of the production of superoxide. This ®c factors able to decrease or increase the activity of production is expected to occur when reduced com- UCP2 and/or UCP3 is of crucial importance to ensure ponents of respiratory chains donate electrons to that changes observed in the coupling state of mito- oxygen, mild uncoupling re-oxidizes respiratory chondrial preparations are due to the activity of these chains and diminishes oxygen concentrations. proteins. Our initial studies with yeast mitochondria showed that high ATP concentrations induce a proton leak- age. On the other hand, phosphate and ADP inhibit this leak.17 Therefore, yeast mitochondria might Expression of UCP1 in yeast adjust the quality of their energy conservation according to the ATP=ADP ‡ Pi ratio. Many years The use of site directed mutagenesis for the study of before phosphate was described as a factor improv- UCP1, made the establishment of suitable expression ing the quality (coupling state) of yeast mitochon- systems necessary. For several reasons, the yeast dria.18 This illustrates how experimental conditions Saccharomyces cerevisiae was chosen by different known to in¯uence the `quality' of mitochondrial laboratories.11,12 It was demonstrated that when preparations may hide processes of possible physio- produced in this yeast, UCP1 was functional in logical relevance. In experiments with yeast mito- mitochondria.11 Moreover, the puri®cation and recon- chondria, a phosphate concentration able to maintain stitution of UCP1 was feasible.12 Afterwards it was this yeast uncoupling pathway inhibited has to be shown that ¯ow cytometry and ¯uorescent potential used. Obviously this has to be taken into account sensitive probes could discriminate between mutants when examining the activity of the new UCP1 of the UCP1.13 Also a correlation was shown between homologs in yeast mitochondria. the growth rate of yeast and the uncoupling power of There are many articles considering the uncoupling several mutants of the UCP1.14 Therefore, these effect of weak acids like fatty acids on mitochondria experiments emerged as possible alternatives to the (these are reviewed in Ref. 19). Accordingly, the tedious preparation of yeast mitochondria. They could uncoupling effects of these molecules require the not give as much information as experiments with presence of proteins, namely members of the mito- yeast mitochondria or reconstitution experiments, but chondrial anion carrier family such as the ADP=ATP as least they could give a ®rst evaluation of the translocator. The model explaining this effect requires uncoupling power of UCP1 mutants, and also have the cycling of FA in the inner membrane: the anionic the advantage of referring to the activity of mitochon- form uses the protein to pass dria in situ. through, and the protonated form moves in the lipid phase. This model has been proposed to also explain the activity of UCP1.20 This is still subject to con- troversy, because the concentrations of FA used to Pathways for protons in `normal induce this cycling phenomenon, were much higher mitochondria' than the one able to activate proton transport by UCP1. Other arguments also suggest that UCP1 is not using the FA cycling phenomenon,21 but is a As we have said before, bioenergeticists took great genuine proton transporter.22 In this respect UCP1 care in isolating ®nely coupled mitochondria. How- looks unique and the physiological relevance of the ever, it appeared that the `non productive respiration' uncoupling due to FA cycling remains uncertain. occurring when substrates are present (state 4) was Therefore, there is no doubt that some mechanisms somehow regulated, and may have some physiological exist to ensure that mild uncoupling of respiration. relevance.15 Accordingly, some mitochondrial uncoup- Unfortunately, the actors responsible for this uncoup- ling may be useful and is not just a waste of energy. ling are poorly characterized. This was well known in plants, where the alternative oxidase of mitochondria, reoxidize coenzymes with- out pumping protons. This results in an uncoupled respiration. Its thermogenic power is used in ¯ow- ers,16 but its main activity is probably to maintain a UCP2 and UCP3 proper redox balance of coenzymes when ATP is coming from chloroplasts. UCP1 in BAT and the alternative oxidase, indicate that the `non productive Pure genetics, or various considerations on the accu- respiration' originates from either leaks through the mulation of artefacts with cDNA and immunological inner membrane or from modi®cations of the respira- probes for UCP1, led to the discovery of UCP2 and tory chains. The bene®ts proposed, (for review see UCP3. The similarity of UCP1 and UCP2 sequences Ref. 15) are: (i) thermogenesis; (ii) the maintenance of associated to the bioenergetic background mentioned a suf®cient respiratory rate of kinetic reasons; (iii) the beforehand1 led us to propose that: UCP2 is a candi- control of redox balance to allow biosynthesis and; (iv) date to explain this leak. UCPs as uncouplers F Bouillaud S21 The expression of UCP3 restricted to muscle made distribution.1 This probably re¯ects two different it an attractive candidate for some regulated thermo- effects of the UCPs expression: A shift towards genesis in mammals.2,4,23 Unfortunately, the two lower ¯uorescence levels of the main population, genes are so close together that it was impossible by indicates a lower capacity of mitochondria to accu- genetics to discriminate their effects. However, a mulate the potential sensitive probe. A second popu- strong association between genetic markers in the lation with very low ¯uorescence value, very likely vicinity of UCP2 and UCP3 genes and resting meta- indicates the appearance of petites mutants, where no bolic rate (RMR),24 reinforced the hypothesis of their more ef®cient mitochondria are found. This is role in its regulation. The changes observed in the expected to be a consequence of the expression of a expression levels of the mRNAs for UCP2 and UCP3, protein that diminishes mitochondrial ef®ciency. gave arguments in favour or inversely against this hypothesis.25 ± 27 An increase in the levels of UCP2 and UCP3 mRNAs seems associated with the use of lipids (highly reduced substrates).1,28,29 It is an over- Isolated mitochondria statement to pretend that cloning of UCP2 and UCP3 was the discovery of the genes determining the basal When mitochondria containing a UCP were compared metabolic rate (BMR). The role of these proteins as to control mitochondria, we noticed a modi®cation of uncouplers of respiration is contested by several respiratory control ratio consistent with the proposal authors. However, we have obtained convincing that mitochondria were partially uncoupled (Rial et experimental data demonstrating the uncoupling al 30). This was well observed with UCP1, and is also effect of the recombinantly expressed UCP2. the case (but to a lesser extent) with UCP2. The situation encountered with UCP3 was more complex and its interpretation was not straightforward. In these UCP2 and UCP3 in yeast studies a non speci®c effect of over-expression might also be proposed. There are several arguments to rule it out: ®rstly the expression is not so high and the There is considerable experience in the study of the recombinant protein is hardly seen in coomassie UCP1 by recombinant expression, and therefore the stained gels of mitochondrial proteins. Moreover same approach was applied to UCP2 and UCP3. measurement of the GDP binding to mitochondria UCP2 and UCP3 were compared to UCP1 with from yeast expressing UCP1 suggests that the respect to their effects on yeast phenotype, before amount present is two or three times lower than in experiments were made with isolated mitochondria. brown fat mitochondria (Rial, personal communica- tion). Secondly, when UCP1 was fully inhibited in the presence of albumin and GDP, `UCP1 mitochondria' were as coupled as control mitochondria (Rial, Gou- Speci®c effect on growth yield bern and Bouillaud, unpublished results). Therefore, the presence of the recombinant protein was function- Expression of UCP2 or UCP3, increased the genera- ally undetectable in these conditions, and this con- tion time of yeast as UCP1 did. This could be ®rmed the lack of a general disturbance of the considered as a non speci®c effect of recombinant mitochondrial inner membrane. This demonstrates expression. This is unlikely, since there are mutants of that when appropriate inhibitors are present, no UCP1, which are expressed as well as the wild type uncoupling effect of UCP1 occurs. Therefore it is of without alteration of the growth rate. Moreover, the critical importance to look for molecules that interact expression of the short form of the (UCP3S)2, induces with UCP2 and UCP3, and change their activity in a very modest increase of the generation time of yeast. isolated mitochondria, but also to examine if the intracellular conditions in vivo allow the uncoupling effect to take place. In this respect methods using whole cells (or organisms) are useful, even if they This effect is mitochondrial cannot lead to `biochemical' conclusions.

In yeast cells expressing UCP1, UCP2 or UCP3, there is an overall decrease in the staining by the potential The key: regulators of uncoupling sensitive probe 3,30 dihexyloxacarbocyanine (DiOC- activity (6)3) in comparison with the control. Separate tests con®rmed that DiOC(6)3 behaves like a mitochon- drial membrane potential sensitive probe in yeast, Although this has been initially proposed, to our since cyanide, antimycine or FCCP decreased the knowledge, there is no convincing evidence of a staining whereas oligomycin increased it (data not regulation by FA and nucleotides of UCP2 or UCP3, shown). The histograms often present a bimodal and we think that UCP2 and UCP3 are not regulated UCPs as uncouplers F Bouillaud S22 like UCP1. However, we have found a natural 9 Enerback S, Jacobsson A, Simpson EM, Guerra C, Yamashita compound and synthetic analogues able to increase H, Harper ME, Kozak LP. Mice lacking mitochondrial uncou- 30 pling protein are cold-sensitive but not obese. Nature 1997, the uncoupling activity of UCP2 (Rial et al ). This is 387: 90 ± 94. strong support for the hypothesis of UCP2 being an 10 Jazek P, Garlid KD. New substrates and competitive inhibitors uncoupling protein, although this does not rule out the of the C1-translocating pathway of the uncoupling protein of possibility of another transport activity mediated by brown adipose tissue mitochondrial. J Biol Chem 1990, 265: UCP2. As we said before, a critical issue will be the 19303 ± 19311. 11 Arechaga I, Raimbault S, Prieto S, Levi-Meyrueis C, Zaragoza physiological relevance of this regulated uncoupling P, Miroux B, Ricquier D, Bouillaud F, Rial E. Cysteine activity. residues are not essential for uncoupling protein function. Biochem J 1993, 296: 693 ± 700. 12 Murdza-Inglis DL, Patel HV, Freeman KB, Jezek P, Orosz DE, Garlid KD. Functional reconstitution of rat uncoupling protein following its high level expression in yeast. J Biol Chem 1991, 266: 11871 ± 11875. Conclusions 13 Bouillaud F, Casteilla L, Ricquier D. A conserved domain in mitochondrial transporters in homologous to a zinc-®nger knuckle of nuclear hormone receptors. Mol Biol Evol 1992, There is no doubt that UCP2 when expressed in yeast 9: 970 ± 975. mitochondria exhibits an uncoupling activity that can 14 Gonzalez-Barroso MM, Fleury C, Arechaga I, Zaragoza P, Levi0Meyrueis C, Raimbault S, Ricquier D, Bouillaud F, Rial be modulated by different factors. No de®nitive con- E. Activation of the uncoupling protein by fatty acids is clusions could be drawn in the case of UCP3, and ®rst modulated by mutations in the C-terminal region of the experiments suggest that UCP3S has no uncoupling protein. Eur J Biochem 1996, 239: 445 ± 450. activity. The effect of the molecules activating UCP2 15 Rolfe DF, Brand MD. The physiological signi®cance of is presently under study in animal mitochondria. It is mitochondrial proton leak in animal cells and tissues. 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