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UCP1, UCP2 and UCP3: Are they true uncouplers of respiration? F Bouillaud1*. 1CEREMOD, CNRS, Meudon, France. The thermogenesis in brown adipose ...
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* 1

CEREMOD, CNRS, Meudon, France

The thermogenesis in brown adipose tissue (BAT) is due to the activity of a mitochondrial uncoupling protein (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 genes showing strong similarities with the UCP1 gene and expressed in other tissues raised the possibility that these proteins 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 The ®rst hypothesis proposed with discovery of 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 comparison between UCP2, UCP3 and UCP1.

Coupled mitochondria Isolated mitochondria start to respire as soon as substrates and oxygen are available, and therefore the energy of substrates is lost. Addition of ADP greatly accelerates respiration and an important part of the energy liberated by the oxidation of substrates is stored into ATP. This implies a coupling between oxidation and phosphorylation, and the controlled permeability to protons of the inner membrane is essential for this coupling. The ratio between the respiratory rate in the presence or absence of ADP (respiratory control ratio) indicates how mitochondria are coupled. More coupled mitochondria are considered as better accordingly biochemical procedures were settled to increase the coupling state of mitochondrial preparations, but one can also examine the possibility that some of the biochemical tricks used to improve the coupling state of mitochrondria, could *Correspondence: Dr F Bouillaud, CEREMOD, CRNS, 9 rue Jules Hetzel, 92190 Meudon, France. E-mail: [email protected]

inhibit or mask naturally present uncoupling mechanisms of physiological relevance. An example is BAT mitochondria, where the uncoupling protein UCP1 is responsible for heat production.

The archetype of uncoupling proteins: UCP1 Isolated BAT mitochondria proved to be largely uncoupled, it took some time to be convinced that this uncoupling re¯ected a physiologically relevant thermogenic mechanism, the discovery of regulators of this uncoupling was a key issue, for review see Refs.5,6 Later on reconstitution of UCP17 and recombinant expression8 showed that UCP1 is the explanation for the fatty acid (FA) induced and nucleotide inhibited, uncoupling pathway of BAT mitochondria. Finally, it was observed that KO mice for the ICP1 gene had a defective cold induced thermogenesis and, therefore, are cold intolerant.9 UCP1 activity results in a transport of protons through the mitochondrial inner membrane. The exact mechanism is still not completely understood. There is evidence from the literature that UCP1 is able to transport many other solutes.10 Therefore if UCP1 would have been just sequenced as an `orphan gene', its role as a thermogenic protein would not have been easy to deduce on the basis of biochemical experiments. The physiologically relevant regulators of UCP1 are probably the FA that strongly activate the proton transport by UCP1 (for review, see Ref. 6 A proton conductance dramatically increased by FA, and inhibited by GDP, indicates the presence of a

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functional UCP1.11 Therefore determination of speci®c factors able to decrease or increase the activity of UCP2 and/or UCP3 is of crucial importance to ensure that changes observed in the coupling state of mitochondrial preparations are due to the activity of these proteins.

Expression of UCP1 in yeast The use of site directed mutagenesis for the study of UCP1, made the establishment of suitable expression systems necessary. For several reasons, the yeast Saccharomyces cerevisiae was chosen by different laboratories.11,12 It was demonstrated that when produced in this yeast, UCP1 was functional in mitochondria.11 Moreover, the puri®cation and reconstitution of UCP1 was feasible.12 Afterwards it was shown that ¯ow cytometry and ¯uorescent potential sensitive probes could discriminate between mutants of the UCP1.13 Also a correlation was shown between the growth rate of yeast and the uncoupling power of several mutants of the UCP1.14 Therefore, these experiments emerged as possible alternatives to the tedious preparation of yeast mitochondria. They could not give as much information as experiments with yeast mitochondria or reconstitution experiments, but as least they could give a ®rst evaluation of the uncoupling power of UCP1 mutants, and also have the advantage of referring to the activity of mitochondria in situ.

Pathways for protons in `normal mitochondria' As we have said before, bioenergeticists took great care in isolating ®nely coupled mitochondria. However, it appeared that the `non productive respiration' occurring when substrates are present (state 4) was somehow regulated, and may have some physiological relevance.15 Accordingly, some mitochondrial uncoupling may be useful and is not just a waste of energy. This was well known in plants, where the alternative oxidase of mitochondria, reoxidize coenzymes without pumping protons. This results in an uncoupled respiration. Its thermogenic power is used in ¯owers,16 but its main activity is probably to maintain a proper redox balance of coenzymes when ATP is coming from chloroplasts. UCP1 in BAT and the alternative oxidase, indicate that the `non productive respiration' originates from either leaks through the inner membrane or from modi®cations of the respiratory chains. The bene®ts proposed, (for review see Ref. 15) are: (i) thermogenesis; (ii) the maintenance of a suf®cient respiratory rate of kinetic reasons; (iii) the control of redox balance to allow biosynthesis and; (iv)

diminution of the production of superoxide. This production is expected to occur when reduced components of respiratory chains donate electrons to oxygen, mild uncoupling re-oxidizes respiratory chains and diminishes oxygen concentrations. Our initial studies with yeast mitochondria showed that high ATP concentrations induce a proton leakage. On the other hand, phosphate and ADP inhibit this leak.17 Therefore, yeast mitochondria might adjust the quality of their energy conservation according to the ATP=ADP ‡ Pi ratio. Many years before phosphate was described as a factor improving the quality (coupling state) of yeast mitochondria.18 This illustrates how experimental conditions known to in¯uence the `quality' of mitochondrial preparations may hide processes of possible physiological relevance. In experiments with yeast mitochondria, a phosphate concentration able to maintain this yeast uncoupling pathway inhibited has to be used. Obviously this has to be taken into account when examining the activity of the new UCP1 homologs in yeast mitochondria. There are many articles considering the uncoupling effect of weak acids like fatty acids on mitochondria (these are reviewed in Ref. 19). Accordingly, the uncoupling effects of these molecules require the presence of proteins, namely members of the mitochondrial anion carrier family such as the ADP=ATP translocator. The model explaining this effect requires the cycling of FA in the inner membrane: the anionic form uses the mitochondrial carrier protein to pass 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 controversy, because the concentrations of FA used to induce this cycling phenomenon, were much higher 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 genuine proton transporter.22 In this respect UCP1 looks unique and the physiological relevance of the uncoupling due to FA cycling remains uncertain. Therefore, there is no doubt that some mechanisms exist to ensure that mild uncoupling of respiration. Unfortunately, the actors responsible for this uncoupling are poorly characterized.

UCP2 and UCP3 Pure genetics, or various considerations on the accumulation of artefacts with cDNA and immunological probes for UCP1, led to the discovery of UCP2 and UCP3. The similarity of UCP1 and UCP2 sequences associated to the bioenergetic background mentioned beforehand1 led us to propose that: UCP2 is a candidate to explain this leak.

UCPs as uncouplers F Bouillaud

The expression of UCP3 restricted to muscle made it an attractive candidate for some regulated thermogenesis in mammals.2,4,23 Unfortunately, the two genes are so close together that it was impossible by genetics to discriminate their effects. However, a strong association between genetic markers in the vicinity of UCP2 and UCP3 genes and resting metabolic rate (RMR),24 reinforced the hypothesis of their role in its regulation. The changes observed in the expression levels of the mRNAs for UCP2 and UCP3, 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 overstatement to pretend that cloning of UCP2 and UCP3 was the discovery of the genes determining the basal metabolic rate (BMR). The role of these proteins as uncouplers of respiration is contested by several authors. However, we have obtained convincing experimental data demonstrating the uncoupling effect of the recombinantly expressed UCP2.

UCP2 and UCP3 in yeast There is considerable experience in the study of the UCP1 by recombinant expression, and therefore the same approach was applied to UCP2 and UCP3. UCP2 and UCP3 were compared to UCP1 with respect to their effects on yeast phenotype, before experiments were made with isolated mitochondria.

Speci®c effect on growth yield Expression of UCP2 or UCP3, increased the generation time of yeast as UCP1 did. This could be considered as a non speci®c effect of recombinant expression. This is unlikely, since there are mutants of UCP1, which are expressed as well as the wild type without alteration of the growth rate. Moreover, the expression of the short form of the (UCP3S)2, induces a very modest increase of the generation time of yeast.

This effect is mitochondrial In yeast cells expressing UCP1, UCP2 or UCP3, there is an overall decrease in the staining by the potential sensitive probe 3,30 dihexyloxacarbocyanine (DiOC(6)3) in comparison with the control. Separate tests con®rmed that DiOC(6)3 behaves like a mitochondrial membrane potential sensitive probe in yeast, since cyanide, antimycine or FCCP decreased the staining whereas oligomycin increased it (data not shown). The histograms often present a bimodal

distribution.1 This probably re¯ects two different effects of the UCPs expression: A shift towards lower ¯uorescence levels of the main population, indicates a lower capacity of mitochondria to accumulate the potential sensitive probe. A second population with very low ¯uorescence value, very likely indicates the appearance of petites mutants, where no more ef®cient mitochondria are found. This is expected to be a consequence of the expression of a protein that diminishes mitochondrial ef®ciency.

Isolated mitochondria When mitochondria containing a UCP were compared to control mitochondria, we noticed a modi®cation of respiratory control ratio consistent with the proposal that mitochondria were partially uncoupled (Rial et al 30). This was well observed with UCP1, and is also 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 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 recombinant protein is hardly seen in coomassie stained gels of mitochondrial proteins. Moreover measurement of the GDP binding to mitochondria from yeast expressing UCP1 suggests that the amount present is two or three times lower than in brown fat mitochondria (Rial, personal communication). Secondly, when UCP1 was fully inhibited in the presence of albumin and GDP, `UCP1 mitochondria' were as coupled as control mitochondria (Rial, Goubern and Bouillaud, unpublished results). Therefore, the presence of the recombinant protein was functionally undetectable in these conditions, and this con®rmed the lack of a general disturbance of the mitochondrial inner membrane. This demonstrates that when appropriate inhibitors are present, no uncoupling effect of UCP1 occurs. Therefore it is of critical importance to look for molecules that interact with UCP2 and UCP3, and change their activity in 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 cannot lead to `biochemical' conclusions.

The key: regulators of uncoupling activity Although this has been initially proposed, to our knowledge, there is no convincing evidence of a regulation by FA and nucleotides of UCP2 or UCP3, and we think that UCP2 and UCP3 are not regulated

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like UCP1. However, we have found a natural compound and synthetic analogues able to increase the uncoupling activity of UCP2 (Rial et al 30). This is strong support for the hypothesis of UCP2 being an uncoupling protein, although this does not rule out the possibility of another transport activity mediated by UCP2. As we said before, a critical issue will be the physiological relevance of this regulated uncoupling activity.

Conclusions There is no doubt that UCP2 when expressed in yeast mitochondria exhibits an uncoupling activity that can be modulated by different factors. No de®nitive conclusions could be drawn in the case of UCP3, and ®rst experiments suggest that UCP3S has no uncoupling activity. The effect of the molecules activating UCP2 is presently under study in animal mitochondria. It is likely that the effect of disruption of these genes in mice will be known well before a satisfying description of their biochemical activities will be provided. The indications given by the phenotype (if any) of these mice and the biochemistry of their mitochondria will be of outstanding importance. Whatever the result, the recruitment of the mechanisms used by mitochondria to change their coupling state remains a possibility of increasing energy expenditure in man.

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