Seminar in Biochemistry
Seminar in Biochemistry MCB 138
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Biochern J 2000 345 161479 Printed in Great Britain 151 REVIEW ARTICLE The uncoupling protein homologues UCP1 UCPZ UCP3 StUCP and AtUCP Daniel RICQUIERl and Frederic BOUILLAUD Centre de Recherche sur l Ehdocrihologie Moleculaire et le Developperneht CEREMOD Centre National de la recherche Scientitigue CNRS 7 Ulil19078 9 rue Jules Hetzel 92i90 Meudon France Animal and plant uncoupling protein UCP homologues form a subfamily of mitochondrial carriers that are evolutionarily re lated and possibly derived from a protonanion transporter ancestor The brown adipose tissue BAT UCPl has a marked and strongly regulated uncoupling activity essential to the maintenance of body temperature in sin ammals UCP 1 1 quot i in 1 i 139 an 39 and may be involved in resistance to chilling The biochemical activities and biological functions of the recently identi ed mammalian UCP2 and UCP3 are not well known However recent data support a role for these UCPs in State 4 respiration respiration uncoupling and proton leaks in mitochondria More over genetic studies suggest that UCP2 and UCP3 play a part in energy expenditure in humans The UCPs may also be involved in adaptation of cellular metabolism to an excessive supply of substrates in order to regulate the ATP level the NADNADH ratio and various metabolic pathways and to contain superoxide production A major goal will be the analysis of mice that either lack the UCP2 or UCP3 gene or overexpress these genes Other aims will be to investigate the possible roles of UCP2 and UCP3 in response to oxidative stress lipid peroxidation in ammatory processes fever and regulation of temperature in certain speci c parts of the body Key words carrier energy mitochondria proton thermogen esis INTRODUCTION This article reviews data on the brown adipose tissue BAT uncoupling protein 1 UCPl two recently discovered mam 39 P homologues UCP2 and UCP3 and plant UCP homologues identi ed in Salanum tuberasum StUCP and Arab idapsis thaliana AtUCP Other review papers on BAT mito chondria or UCPs have been published in recent years 1715 Two other carriers predominantly expressed in brain and showing some uncoupling activity in recombinant systems were recently identi ed and referred to as BMCPl brain mitochondrial carrier protein 1 l6 and UCP4 17 These two carriers are less similar to the UCPs and will not be considered in this review Uncoupling ol respiration Mitochondrial ATP synthesis from ADP driven by electron ow from reduced substrate mainly NADH to oxygen de nes oxidative phosphorylation Peter Mitchell proposed that oxid ation is coupled by the electron transport chain to pumping of protons from the mitochondrial matrix generating a proton motive force or electrochemical potential difference for protons across the inner membrane of the mitochondria This force drives the protons back into the matrix through ATP synthase which couples proton transport across the membrane to phos phorylation of ADP 18 The observation that mitochondria still consume oxygen when ADP phosphorylation is inhibited demon strates that the coupling of respiration to ATP synthesis is imperfect in fact State 4 respiration is mainly due to uncoupling 1920 The coupling of oxidative phosphorylation is impaired by the existence of certain leaks through the mitochondrial inner membrane 19726 Mechanism ol respiration uncoupling proton leaks versus slip The existence of two types of mechanism for respiration un coupling has been debated for several years for a review see 27 On the basis of Mitchell s theory it was proposed that an increased proton permeability of the mitochondrial inner mem brane not coupled to an energyconsuming system such as ATP synthase would constitute a proton leak and decrease the level of coupling of respiration to ADP phosphorylation An alterna tive explanation of uncoupling corresponds to the concept of slip The slippage of respiratory chains can be de ned as a failure of the proton pumps of the respiratory chain which transfers electrons without extruding protons out of the mem brane The slippage of respiratory chains implies that a higher respiratory rate is necessary for the predicted ATP synthesis Biological roles and henelils ol respiration uncoupling Besides the biochemical mechanisms of respiration coupling uncoupling the biological signi cance of such a mechanism can be considered Leaks and slips which explain the partial ef ciency of energy transduction through the mitochondrial membrane favour wastage of free energy but may also have functional roles bene cial to organisms 22 A major consequence of the un coupling of respiration is the activation of substrate oxidation and dissipation of oxidation energy as heat In terms of physi ology uncoupling of respiration and dissipation of energy as heat are obviously important for ener y balance and bod duction due to uncoupling are precisely observed when the BAT UCPl is working in animals exposed to the cold in newborn mammals or when hibernators are waking from hibernation ln AbbreViations used Alli activator proteinril BAT brown adipose tissue DlT dietrinduced therrnogenesisl ROS reactive oxygen species UCP uncoupling protein AtUCP UCP homologue in Arabldopsls thallanal StUCP UCP homologue in Solanum tuberosum To whom correspondence should be addressed eyrnail ricquierintobiogen tr 2000 Biochemical Society 162 D Rlcqmer and F BomHaud other respects in addition to a contribution to the basal metabolic rate and adaptive thermogenesis it was proposed that mild uncoupling of respiration could prevent the accumulation of oxygen radicals generated by mitochondria 28 Rolfe and Brand 20 have proposed that partial uncoupling may increase the sensitivity and rate of response of oxidative phosphorylation to effectors Another role for uncoupling of respiration is to control the NADtNADH ratio and regulate metabolic pathways such as ketogenesis lipogenesis and amino acid synthesis which are dependent on the levels of these coenzymes 2829 BAT UCP1 A MITOCHONDRIAL UNOOUPLING PROTEIN OF AN ENERGYDISSIPATING ORGAN The discovery of the BAT UCP renamed UCP1 when UCP2 was discovered 3031 resulted from studies of the mechanism ofthermogenesis in BAT The thermogenic function ofBAT was demonstrated around 196171964 by several groups working on rodents or lagomorphs These groups reported that BAT pro duces heat in particular under conditions requiring extra heat production such as exposure to the cold birth or arousal from hibernation for reviews see 17393233 BAT is present in almost all mammals It is a major site of coldinduced thermogenesis in rodents and also contributes to dietinduced thermogenesis DlT 34 This tissue is well de veloped around birth in large mammals and throughout the lifespan in rodents BAT is found in characteristic deposits scattered in speci c areas in the body the major deposits being interscapular axillar perirenal thoracic and between the neck muscles The topology of BAT is such that upon activation of brown adipocytes heat is quickly cleared through large vessels which convey it to the thoracic spinal chord heart thoracic structures brain and kidneys BAT is composed of brown adipocytes which differ from white adipocytes Brown adipocytes contain several droplets of triacylglycerols a central nucleus and many mitochondria In addition the mitochondria are striated and exhibit numerous cristae generated by the highly developed inner membrane Morphological analysis of brown adipocytes suggested that these cells have a marked oxidative capacity and this was con rmed by measurements of oxygen consumption of isolated brown adipocytes The role of BAT in regulatory thermogenesis implies that its activity is regulated The main control system of BAT activity is located in the hypothalamus and acts through thermoregulatory centres and sympathetic nerves which innervate the brown adipocytes directly Activation of BAT thermogenesis by sym pathetic bres and noradrenaline was demonstrated in studies showing that noradrenaline released at the surface of brown adipocytes activates adrenoceptors increasing oxygen consump ti n and inducin heat production wi hin 1 or 2min after quot rIPIix pm Since quot arh39x aha lipolysis it was proposed that the increased oxidation of fatty acids was related to raised heat production Although increased respiratory activity of BAT mitochondria constitutes a thermogenic process per Se the existence of a particular mechanism operating in these mitochondria was suspected from 1967 when Robert Smith and Olov Lindberg and their collaborators independently observed that the res piration of mitochondria isolated from BAT was loosely coupled to ADP phosphorylation for reviews see 12 Several re searchers then demonstrated that the uncoupled respiration of BAT mitochondria was activated by fatty acids and inhibited by purine nucleotides such as GTP GDP ATP or ADP 12 These data suggested the existence of a regulatable uncoupler in the inner membrane of BAT mitochondria 2000 Blochemlcal Somety Although it is not known if regulation of the uncoupling pathway by the binding and debinding of nucleotides is physio logical the activation of uncoupling by fatty acids has a strong physiological signi cance since activation of BAT by nor adrenaline obligatorily triggers lipolysis A major breakthrough was made by David Nicholls and his collaborators when they measured a particularly high proton conductance of BAT mitochondria and observed that this proton pathway was inhibited by GDP and activated by fatty acids Using photo ai nity labelling experiments they identi ed a 32kDa protein as the binding site of nucleotides and the putative UCP 35 Before the publication of these data a 32kDa protein abundant in the membranes of BAT mitochondria was described this pro tein was absent from liver mitochondria induced during exposure of rats to the cold for several days and downregulated after re adaptation to room temperature 36 Following these studies the 32kDa protein was puri ed from hamster and rat 3738 sequenced 39 cloned as cDNAs 40413 and its proton translocating activity reconstituted in liposomes 44417 It was therefore accepted that this protein was responsible for the regulated loose coupling of BAT mitochondria and it was generally referred to as the uncoupling protein UCP UCP1 Other authors referred to this protein as thermogenin 2 Finally the demonstration of an important role for UCP1 in adaptive thermogenesis was provided by Enerback et al 48 who obtained null mutant mice unable to maintain their body temperature in a cold environment UCP1 synthesis is strongly activated by noradrenaline cAMP thyroid hormones and retinoids for reviews see 289 Dietary vitamin A supplementation in rats 49 or the addition of natural carotenoids to brown adipocytes induces UCP1 50 Chronic treatment of obese rats by nicotine was reported to induce UCP1 in white fat depots 51 IDENTIFICATION OF UCP HOMOLOGUES UCPZ UCP3 AND PLANT UCPS The speci c expression of UCP1 in brown adipocytes has been con rmed in many laboratories see reviews cited in the ln troduction section Nevertheless the molecular mechanisms of partial coupling of respiration in mitochondria of most tissues remained unclear The debate between advocates of proton leaks and supporters of slippage of respiratory chain remains open although it seems reasonable to assume that the two types of mechanism exist 52 The existence of proton leaks accounting for 20730 of total oxygen consumption of liver or skeletal muscle mitochondria 2122 indicates that certain components of the inner membrane of mitochondria catalyse proton leaks Brookes et al 53 recently concluded that the mitochondrial proton leaks are not due to phospholipids Since the BAT UCP uan pun plutuus through theiuuei f 39 39 it was tempting to propose that UCP homologues were trans locating protons through the inner membrane of liver or skeletal muscle mitochondria In fact over the last few years we have observed that antibodies against UCP1 or UCP1 cDNAs are occasionally able to bind to protein or RNA from tissues other than BAT Whether such interactions are due to nonspeci c binding to protein or RNA of other known mitochondrial carriers or UCP1 homologues had not been investigated In addition the complete sequencing of SaccharamyCes Cereuisiae yeast revealed the putative existence of 34 proteins related to the amily of mitochondrial carriers 54 suggesting that this family was much larger than expected Taken together these data encouraged us to look for UCP homologues expressed in different tissues of mammals and to Uncoupluo prmelns lea Fhlm I thous Mannm analysts at ucPIucP1 and um mums Iu mous Pumuroolzuoo mml RNMmmeszssue oxmm an lung we room mm on Tms Flume was kuoly women by ur namel saucms clone 1ch a UCPl homologue present rn several ossues of mrce and humans whrch rs 59 rdenocal to UCPl 30 ucpz lhr n or reversetranscnptasePCRanalysrs 3155 Athrrd ucpreterred to as ucpz and shanng 72 rdenuty wrth ucpz ms rdenu ed by several groups a tew mont s alter the drscovery of U 2 55759 Frgure 1 shows the drstrrbuuon of UCPl ucpz and l t l r m tel descrrbed by Mao et al 17 who proposed to name rt UCPA although the ammo acrd rdenuty wrth the UCPs rs only around 30 BIOCHEMISTRV OF THE UCPs Primary and secondary slmmnzs The alrgnment of the ammo acrd sequences of UCPl ucpz and ucpz shows that these proterns are srgnr cantly srmrlar see a 2 E a 5 a a S 3 0 as a 5 3 P sonrcated mrtoplasts These studres valrdated the predrcted s ondary structure otucpl Although no other data are avarlable rt ca of toldrng rn the membrane Immunological Maniaquot ol UcPs There are tew reports descnbrng specr c and sensruve anobodres agarnst mrtochondnal carners such as the ADPATP camer r t t th rer almost no convrncrng publrshed descnpuon of anobo reacong wrth ucpz or ucpz whereas anu bodres agarns usablernrmmunohrstochernrstry Western analysrs an Immunor precrprtaoon expenments were obtarn e exU rn 1997 the clonrng of the rst plant ucp present rn Solavmm tubevomm and reterred to as StUCP cDNAs of a ucp homr ologue present rn Ambzdopm mm were a so cloned and reterred to as Atucp cDNA 51 lnteresongly usrng anubodres agarnst plant uc Jarmuszlnewrcz et al 52 detected a mum chondrral protern rn the nonrphotosyntheuc sorl amoebord moeba cccazzcmc ma provrdeanetp n ontorthemrtochondnalprotonleaks Whether such enes encode ns th rde o l lated o drsonct properoes rs drscus e rs the contnbuoon s h re as of these proterns to mrtochondnal proton leaks The quesoon of k thedrmcultyrnrdenotyrng UCPZo mr rs due to very low levels of ucpz or ucpz rn ossues or to the whrch onotexpxessUC lor ressuc Zm suggesun that the anogen was n the same study Western analysrs ms d to how that anurUCPl anubodres could react wrt ucpz overexpress rn yeast mrto ndna Usrn the same anurUCPl nuser hr h expressron of a and macrophages present rn toetal lrver 71 and the level of couplrng of resprraoon rs rn tact complrcated rw TD carner and aspartateglutamate anoporter catalyse a proton transport acovated by tatty acrds 5mm ln other respects rn the course of expenments on UCPl etpressron rn yeast carned ou collaboraoon wrth Eduardo Rral a proton transport acuvrty acovated byA r named the yeast uncouplrng pathway or YUP 57 A new 5 esom srnce toetal lrver contarns ucpz mRNA whereas UCPl and ucpz mRNAs are undetectr M U P r adult mrce Chavrn et al 72 used an anoserum drrected the Nrmmnzl extremrty of uc not detect a hrgh level o an gen rn t e rvers otcon ro mrce whereas a srgnr cantrnducoon otthe protern was o serv Northern analysrs Two groups recently reported the detecuon of UCPZ rn 74 brarn and reterred to as BMCPI carner protern l was also ldenu ed la Cunously although the se quence of thrs new protern rs only moderately srmrlar to the 4 muscle usrng Western analysrs wrth anubodres drrected erther agarnst the Nrmmnzl 75 or the Crkemnnzl 751extremrty of M marked uncouplrng of resprraoon and proton leak nother mrtochondnal carner expxessad rn brarn and some uncouplrng acuvrty rn mammalran cells ms rnduced a Frnally a showrng A descnpoon of an ucpz anogen rn Western analysrs of rat whrteadrposeossuemsnotenurelyconvrncrngsrncetheauthors uolrzred an anoserum agarnst UCPl and srncewhrtetatcontarns zuuu armtemrml SCEEW 164 D Ricquier and F Bouillaud UCP MGGLTAS DVHPTLGVQLFSAGIAACLADVITFPLDTAKVRLQVQGECP TSSVIRYKGVLGTITAVVKTEGRMKLYSGLPAGLQRQISSASLRIGLYDTVQEFL UCP2 MVGFKAT DVPPTATVKFLGAGTAACIADLITFPLDTAKVRLQIQGESQGPVRATASAQYRGVMGTILTMVRTEGPRSLYNGLVAGLQRQMSFASVRIGLYDSVKQFY UCP3 MVGLKPS DVPPTMAVKFLGAGTAACFADLVTFPLDTAKVRLQIQGENQ AVQTARLVQYRGVLGTILTMVRTEGPCSPYNGLVAGLQRQMSFASIRIGLYDSVKQVY U CP1 TAGKETAPS LGSKILAGLTTGGVAVFIGQWAQSHLHGIKP RYTGTYNAYRI IATTEGLTGLWKGTT PNLMRSVI INCTELVTYDLMKEAFVKN U CP2 TKGSE HAS IGSRLLAGSTTGALAVAVAQPTDWKVRFQAQARAGGGR RYQSTVNAYKTIAREEGFRGLWKGTSPNVARNAIVNCAELVTYDLIKDALLKA U CP3 TPKGADNSS LTTRI LAGCTTGAMAVTCAQPTDWKVRFQASIHLGPSRSDRKYSGTMDAYRTIAREEGVRGLWKGTLPNIMRNAIVNCAEVVTYDILKEKLLDY k k U CP1 NILADDVPCHLVSALIAGFCATAMSSPVD KTRFINSPPGQYKSVPNCAMKVFTNEGPTAFFKGLVPSFLRLGSWNVIMFVCFEQLKRELSKSRQTMDCAT U CP2 NLMTDDLPCHFTSAFGAGFCTTVIAS PVDWKTRYMNSALGQYSSAGHCALTMLQKEGPRAFYKGFMPSFLRLGSWNVVMFVTYEQLKRALMAACTSREAPF UCP3 k HLLTDNFPCHFVSAFGAGFCATVVASPVDVVKTRYMNSPPGQYFSPLDCMIKMVAQEGPTAFYKGFTPSFLRLGSWNVVMFVTYEQLKRALMKVQMLRESPF kkk k k Figure 2 Alignment of UCP1 UCP2 and UCP3 amino acid sequences showing the triplicated structure of mitochondrial carriers Sequences of the human proteins are given The signatures of mitochondrial carriers are underlined and shaded Identical and similar 2 amino acid residues are indicated Table 1 Molecular masses M in Da and the numbers of amino acid aa residues n were calculated from proteins without the initiation methionine mUCP mouse UCP rUCP rat UCP hUCP human UCP Skm skeletal muscle Two forms of human UCP3 were predicted from cDNA cloning experiments a long form UCP3L and a short form UCP3SI The level of identity corresponds to the same amino acid residues at the same positions Most data presented were calculated using sequence data in GenBank Main characteristics and relative identities of the UCPs Identity UCP MDa AA n Distribution With UCP1 With UCP2 pl mUCP1 33117 306 BAT 100 56 93 rUCP1 33 080 306 BAT 57 92 hUCP1 32873 306 BAT 57 93 mUCP2 33342 308 All tissues 59 100 98 rUCP2 33 245 308 All tissues 57 97 hUCP2 33098 308 All tissues 59 97 mUCP3 33779 307 8km BAT 54 73 96 rUCP3 33884 307 8km BAT 54 72 96 hUCP3L 34084 311 Sk m 57 72 93 hUCP3S 29651 274 Skm StUCP 32 327 305 All organs 41 43 94 AtUCP 32 531 305 All organs 41 46 96 brown adipocytes 77 Others reported the immunodetection of UCP3 in Western analysis of BAT or gastrocnemius muscle extracts but the immunoblot pictures and competition with antigenic peptides revealed the presence of numerous bands such that the identi cation of UCP3 itself was problematic 78 The detection of UCP2 in mitochondria from rat tissues by a group who used antiUCP3 antibodies was not convincing 79 In conclusion although several immunohistochemical studies have identi ed an antigen probably corresponding to UCP2 there is little evidence for the speci c detection of UCP2 and UCP3 using Western analysis The speci city of the available antibodies remains to be fully demonstrated and only micro sequencing of immunodetected or immunoprecipitated tissue antigen will prove the identity of the proposed UCP2 and UCP3 antigens Mitochondrial localization of the UCPs The subcellular localization of animal and plant UCPs is strongly suggested by their primary structure and the presence of the 2000 Biochemical Society signature of mitochondrial carriers see above although it was reported that certain proteins of this family may be present in peroxisomes 80 In the case of UCPl the localization in mitochondria was demonstrated by functional assays immuno detection and puri cation Moreover the marked uncoupling activity of UCPl overexpressed in yeast 78182 adipose tissue mitochondria 83 and skeletalmuscle mitochondria F Bouil laud A M CassardDoulcier C Fleury D Ricquier and P Diolez unpublished work as well as the analysis of UCPl mice 48 convincingly established the mitochondrial localization of UCPl In the case of UCP2 UCP3 and the plant UCPs fewer data are available However the signi cant effect of these UCPs on the mitochondrial membrane potential of recombinant yeast 303159608485 supported their mitochondrial localization In addition UCP2 and UCP3 were immunologically detected in the mitochondrial fraction of recombinant yeast 7085 Ex pression of UCP2 in COS cells and cellfree synthesis of UCP2 followed by in vitro import experiments demonstrated that this UCP was targeted to mitochondria C L viMeyrueis C Gelly D Sanchis I Cohen C PripsBuus and D Ricquier unpub lished work Finally expression of UCP3 fused to an epitope in human breast carcinoma MCF7 cells demonstrated its subcellular localization in mitochondria 17 Uncoupling mechanism of UCP1 The uncoupled state of BAT mitochondria is explained by an exceptional permeability of the inner membrane to protons and anions 86 related to the presence of UCPl 1 Studies with respiring mitochondria demonstrated that both removal of non esteri ed fatty acids and the presence of nucleotides were necessary to ensure proper recoupling and UCPl inhibition 2 86 Concerning the regulation of UCPl activity most re searchers agree on its inhibition by nucleotides and its activation by nonesteri ed fatty acids a requirement for the carboxy groups of fatty acids the ability of UCPl also to transport various anions the existence of distinct fatty acid and nucleotide binding sites and the identi cation of certain amino acid residues at the nucleotidebinding site However the mechanism of proton transport by UCPl is still debated for reviews see 7131887 The amino acid sequence of UCPl indicates that it is a member of the family of the anion carriers present in the mitochondrial inner membrane The distinction between a carrier and a channel relies on the maximal rate of transport which is much higher in a channel and on the type of interaction between transported molecules and the protein In carriers a more intimate interaction results in a change of conformation of the Uncoupling proteins 165 Figure 3 Mitochondrial Matrix Models of the mechanism of action of UCP1 showing H transport and the participation of fatty acids Shown are a schematic representation in the membrane and a model of proton transport in UCPi in the mitochondrial inner membrane Pink rectangles represent regions of the protein participating in the constitution of the channel domain and which are probably transmembranous ocheliCBS The grey oval represents the gating domain The orientations of the electrical potential and of the ApH component are indicated a b Hypothesis of a proton pathway present in UCPi This pathway may operate in the absence a or presence b of fatty acids The carboxy group of a fatty acid participates in the constitution of the proton pathway and allows or enhances proton transport b this mechanism lowers the membrane potential and therefore increases respiration c Fatty acid cycling hypothesis the anion carrier UCPi allows the completion of a protonophoric cycle of fatty acids in the membrane the fatty acid anion transport is due to the protein whereas the protonated form diffuses freely in the membrane We propose that this mechanism operates when the fatty acid concentration is relatively high d Whatever the mechanism a b or c binding of nucleotide induces a conformational change that makes transport impossible Data suggest that there is no competition between nucleotides and fatty acids for the same site protein that is necessary for transport 88 whereas in a channel ions go through a pore 89 Kinetic analysis of proton transport through UCPl indicated that although a carrier mechanism was suspected it was instead functioning as a channel 90 The study of mitochondrial anion carriers has shown that limited mod i cations can convert carriers into channels These modi cations include manipulation of the ionic composition of the medium in the case of the ADPATP carrier 91 chemical modi cation in the case of the phosphate carrier 92 or sitedirected muta genesis in the case of UCPl 93 Following such modi cations a loss of speci city of the transport was noted and sometimes the channel exhibited certain porelike properties and allowed large molecules to go through This suggested that the anion carriers of the mitochondrial inner membrane contain two distinct domains a channellike structure and a gating domain The involvement of such a shift between carrier and channel properties was suggested to occur in viva for the ADPATP translocator and would explain the phenomenon referred to as the mito chondrial transition pore As opening of the mitochondrial transition pore is thought to be a primary event in the execution programme of apoptosis 94 the identi cation of mechanisms underlying carrier channel conversion is of great interest The high ionic permeability of BAT mitochondria was assigned to UCPl activity since purine nucleotides and more speci cally GDP could inhibit this leak It was concluded from these pioneer studies that UCPl was able to transport protons and several anions such as chloride bromide and nitrate 86 In later studies additional anions were also found to be transported by UCPl 96 In theory proton transport by UCPl contradicts its relationship with anion carriers However cotransport of a proton by the mitochondrial phosphate carrier ensures electro neutrality 97 or makes the exchange sensitive to the elec trochemical gradient across the inner membrane in case of the aspartate glutamate carrier 98 Therefore proton pathways existed in mitochondrial carriers before the appearance of UCPl in mammals The study of BAT mitochondria from mice de cient in UCPl showed the absence of the GDPsensitive proton leak and con rmed the proton transport function of UCPl 99 Over expression of UCPl in skeletal muscle of mice induced a marked mitochondrial proton leak that was activated by nonesteri ed fatty acids and inhibited by GDP A M CassardDoulcier C Fleury D Ricquier M Goubern and F Bouillaud unpublished work Matthias et al 95 recently analysed the bioenergetics of BAT mitochondria from UCPlablated mice and observed that surprisingly these mitochondria were as sensitive to the un coupling effect of nonesteri ed fatty acids as were UCPl containing mitochondria In contradiction of a well accepted mechanism they concluded that fatty acids do not seem to be the intracellular physiological activator of UCPl There are currently two proposed mechanisms to explain the uncoupling activity of UCPl and the role of fatty acids see Figure 3 In the rst model proposed by Klingenberg 13 UCPl actually transports protons and fatty acids provide an essential free carboxy group making proton transport possible or more efficient Figures 3a and 3b The second model proposed by Garlid 87 involves cycling of fatty acids in the membrane with UCPl ensuring the transport of the anionic form of fatty acids Figure 3c The fatty acid cycling model is supported by experiments carried out with both reconstituted systems and mitochondria For example the inhibition of proton transport when azido fatty acids were immobilized on UCPl indicated that movement of the 2000 Biochemical Society 166 D RiCqUiSi and F Bomiiaud fatty acid is necessary to transport protons under the exper imental conditions used 100 However it is not certain if this model explains UCPl activity in viva Firstly this mechanism was originally proposed to explain the uncoupling effect of fatty acids in mitochondria devoid of UCPl the proteins involved being other anion carriers of the same family and the ADP ATP carrier in particular 101102 This proposal suggests that all the transporters of the mitochondrial inner membrane are potential uncoupling proteins in the presence of fatty acids 6487103 Secondly in the case of UCPl experiments supporting this model were carried out using high concentrations of fatty acids able according to the authors to induce a signi cant partial lysis of mitochondria 104 Therefore although this does not invali date this mechanistic model it is likely that under similar experimental conditions many other carriers would behave as uncoupling proteins 63 In terms of this model the purpose of UCPl gene expression in BAT could be understood in its more extreme interpretation as a mere overloading of the mito chondrial inner membrane with a carrier protein which by virtue of a characteristic common to all these proteins would uncouple respiration when fatty acids are present It still makes sense to consider the possibility that besides the fatty acid cycling mechanism UCPl possesses a speci c pathway for proton transport thus making UCPl different from the other carriers Several arguments can be put forward to support the contention that a proton pathway exists in UCPl and that fatty acid cycling is not absolutely necessary for uncoupling to occur Mitochondria endowed with a variety of other carriers are more coupled than are BAT mitochondria The introduction of UCPl either in CHO cell mitochondria or in yeast mitochondria using recombinant DNA technology induced a partially uncoupled state 81105 When yeast mitochondria containing UCPl were challenged with fatty acids they showed a much higher sensitivity to the fatty acids than the control mitochondria 106 The inhibition of both the basal and the fatty acidinduced uncoupling by nucleotides indicated that the proton transport was due to UCPl 106 As expected addition of a higher concentration of fatty acids partially uncoupled control mitochondria without UCPl but in contrast this uncoupling could not be inhibited by nucleotides 106 Certain modi ed fatty acids with hydrophilic substitutions make the ip op across the hydrophobic phase very unlikely Such modi ed fatty acids were still able to activate proton transport through UCPl 13 These data suggest that cycling is not absolutely required for proton transport to occur In the fatty acid cycling model fatty acids are obligatory for proton ux and uncoupling However the observation ofUCPl mediated uncoupling in the absence of fatty acids would indicate that the proton pathway still operates when fatty acid cycling cannot take place As indicated above data obtained with respiring mitochondria showed that even when fatty acids were trapped by albumin mitochondria were still uncoupled and lowering of the respiratory rate required the further addition of nucleotides 106 The ef ciency of fatty acid removal by albumin has been questioned and therefore remaining nonexchangeable fatty acids have been proposed to explain the persistence of the uncoupling activity of UCPl in BAT mitochondria 87 This possibility is however poorly compatible with the fact that UCPl activation in reconstitution experiments requires relatively high concentrations of fatty acids Moreover the fatty acid cycling model implicates a pool of fatty acids in equilibrium between UCPl and the lipid phase of the membrane and it is therefore dif cult to understand why this fatty acid pool would not equilibrate with albumin Finally in the yeast recombinant expression system the basal rate of proton leak catalysed by UCPl in respiring mitochondria was not in uenced by a mutation 2000 Biochemical Society increasing UCPl sensitivity towards fatty acids 106 These servations suggest that in respiring mitochondria where high and stable membrane potential value was reached UCPl was able to catalyse aproton back ow inducing ahigh uncoupled respiratory rate not dependent on fatty acid activation In conclusion we propose the following hypothesis whereby the mechanism of the uncoupling activity of UCPl changes according to the fatty acid concentration Figure 3 a proton pathway exists in UCPl this pathway is suf cient to generate a partial uncoupling of mitochondria in State 4 of respiration when a high membrane potential is present Figure 3a addition of small amounts of fatty acids greatly increases the proton conductance through UCPl Figure 3b at the same time a fatty acidinduced uncoupling occurs in UCPl as it does in other anion carriers and could be the dominant mode of transport when high concentrations of fatty acids are present Figure 3c binding of nucleotides to UCPl induces a change in conformation that inhibits all of these transport modes Figure 3d Mutagenesis studies and iunciional organization oi UCP1 Using recombinant expression of UCPl in various systems site directed mutagenesis was used to test several hypotheses on the activity and the regulation of UCPl in terms of nucleotide inhibition pHsensitivity of nucleotide binding and proton transport Mutagenesis of single arginine residues abolished the inhibition by nucleotides of transport through UCPl 107 Arginine residues at positions 83 182 and 276 were predicted to be located in the transmembranous ochelices These data sup ported the existence of a pocket in the plane of the membrane where the nucleotide binds When the amino acid sequence of UCPl became available a short region of greater similarity to the ADPATP carrier was noted 42 Subsequently it was observed that this small domain corresponding to amino acid residues 2617269 in rat UCPl was very similar to an ochelix interacting with DNA in nuclear receptors such as the oestrogen receptor 82 This led to the prediction that this region is part of the nucleotidebinding site and of its ochelical arrangement In agreement with this prediction the deletion of amino acids 2677269 resulted in a UCP which could still be activated by fatty acids as expected but was no longer inhibited by nucleotides 82 The binding of nucleotides to UCPl is highly sensitive to pH When the pH rises the af nity of UCPl for nucleotides declines notably above pH 72 108 pH changes have therefore been considered as a possible mechanism ofregulation of UCPl in the brown adipocyte The glutamic acid residue at position 190 has been demonstrated to be essential for the pHsensitivity of nucleotide binding 108109 Mutagenesis of histidine residues in UCPl indicated that a pair of histidine residues His145 and His147 is important for proton transport 110 The two histidine residues were predicted to be located in the second matrix loop of UCPl Whereas proton transport was suppressed mutation of the two histidines had no effect on chloride transport suggesting that the transport mechanism was still intact but that its speci city was changed 110 These data suggested the existence of a proton pathway inside the hydrophilic domain of UCPl involving proton exchanging amino acids and were poorly compatible with the fatty acid cycling model It is notable that these two histidines are not conserved in UCP2 and UCP3 This suggested to Bienen graeber et al 1 10 that a common mechanism of proton transport in UCPl UCP2 and UCP3 was unlikely or that UCP2 was not a proton transporter However these two hypotheses were ruled out i Zhang et al 111 reported that mutagenesis of the two Uncoupiing proteins 167 Table 2 Findings tor and against an uncoupling activity or a role in energy expenditure lor UCPZ UCP3 and StUCP Findings are trom reterences quoted in the text abstract 254 i F Bouiiiaud and M Goubem unpubiisned work Roie For Against Uncoupiing actiyity 1075910 amino acid identity with UCPt Decrease in mitocnondnai membrane potentiai in recombinant yeast expressing UCPZ UCPS iant UCPs orp increase in respiratory rate and decreased respiratory controi ratio in recombinant UCPZ or UCPS yeast Amounts 0t mRNAs do not match mitochondriai i39i r conductance No UCP detected in hepatocytes 0t normai rodents Uprreguiation 0t mRNAs in starvation is not associated with increased i39i r conductance increase in proton ieailt in UCPZ spneropiasti39 Proton transport actiyity 0t UCPZ and UCPS in iiposomes Roie in energy expenditure Uprreguiation 0t UCPZ and 00 in m inermogenesis in yeast expressing UCPZ or UCPS P mRNAs i e and piaan exposed to the coid Uprreguiation 0t mRNAs by tnriodotnyronine is Uprreguiation 0t mRNAs by pyretic compounds No increase in UCPS mRNA expression in coidrexposed animais Uprreguiation in skeietai muscies atter starvation Uprreguiation atter one bout 0t exercise Correiation 0t mRNA ieyei witn resting energy expenditure Strong genetic iiniltage to resting metaboiic rate in humans Association 0t gene poiymorpnisms witn resting energy expenditure No association 0t gene poiymorpnisms witn resting energy expenditure histidine residues in UCPl did not impair its ability to uncouple respiration in yeast mitochondria and ii Jaburek et al 112 demonstrated that UCP2 and UCP3 catalysed the electrophoretic ux of protons in liposomes The complete deletion of the domain formed by residues 2617269 in UCPl generated a mutant protein with porelike properties since ionic speci city was lost and solutes up to 1000 Da in molecular mass could pass through 93 This short region was predicted to be localized at the extremity of the third hydrophilic loop and therefore to constitute the hinge between this domain and the sixth transmembranous ochelix 69 The activity of this mutant supported the existence of a gating domain the destruction of which resulted in the formation of a porelike protein with no speci city and with a cutoff close to that of the mitochondrial transition pore A 20aminoacid peptide containing residues 2617269 of a matrix loop was analysed by NMR and circular dichroism 113 These studies con rmed that matrix loops contribute to the gating domain in P1 Proton transport activity at UCPZ UCP3 and plant USPS and regulation by ligands A limited number of analyses of the biochemical activity of the novel animal and plant UCPs are available The present data indicate that UCP2 UCP3 StUCP and AtUCP alter the mitochondrial membrane potential recorded in viva in recom binant yeast and mammalian cells and probably uncouple respiration 17303157597618485 The respiration ofisolated yeast mitochondria into which UCP2 had been introduced was less coupled than that of control mitochondria 30 The portion of cellular respiration coupled to oxidative phosphorylation was decreased in yeast overexpressing UCP3 85111 Increased ther39mogenesis was recorded in yeast overexpressing either UCP2 114 or UCP3 85 Interestingly hepatocytes of mutant obese 017017 mice express UCP2 whereas hepatocytes of 017 mice do not 72 Comparison of the proton leak in liver 39 from these two strains of mice revealed an increased proton permeability in the liver mitochondria of 017017 mice in which UCP2 is present 72 This nding suggested that UCP2 levels are correlated with mitochondrial proton leak A major advance was made by Garlid and his coworkers This team reconstituted the transport activity of human UCP2 and UCP3 puri ed from recombinant bacteria into liposomes and observed that the two proteins catalysed an electrophoretic ux of protons similarly to UCPl 112 In addition the same group reported that fatty acids were obligatory for the proton transport activity of UCP2 and UCP3 again similar to UCPl 112 These data led the authors to conclude that UCP2 and UCP3 are true UCPs having transport properties that are qualitatively identical with those of UCPl with respect to transport of protons and alkylsulphonates112 However using yeast mitochondria containing UCP2 or UCP3 we were unable to measure any activation of the proteins by fatty acids under conditions where a net activation of UCPl was recorded 115 We and others were also unable to inhibit the uncoupling activity of UCP2 and UCP3 by adding nucleotides to isolated mito chondria111115 Interestingly using liposomes Jaburek et al 112 observed that the proton transport activity of UCP2 and UCP3 was not inhibited by low concentrations of nucleotides with such an inhibition being observed only with high levels of nucleotides In contrast Echtay et al 116 reported the re constitution of the chloride transport activity of UCP3 and observed an inhibition by nucleotides such that UCP3 was more sensitive to ADP than to ADP unlike UCPl Retinoids were identi ed as novel regulators of the uncoupling activity of UCPl and UCP2 when tested in yeast mitochondria 115 Using the same experimental conditions these regulators did not affect UCP3 activity expressed in yeast 115 Taken together all these data suggest that the regulation of UCPl UCP2 and UCP3 activity by ligands may somehow differ These in vitr0 experiments supported an uncoupling activity of the novel UCPs but they did not demonstrate that this is the 1 1 main roleof A UCP31 1 2000 Biocnemicai Society 168 D Ricquier and F Bouillaud NAD ApH I Redox RC g H Mitochondria NADH ATP Cell Figure 4 Proposed links between the redox state of coenzymes and ATP production by mitochondria The mitochondrion is depicted as a box Four components are represented the respiratory chain R0 the regenerating oxidized coenzymes the F0F1 ATPase and the transporters of the inner membrane A S UCP These transporters usually exchange anions through antiport A or co translocate anions and protons though symport 8 resulting in electroneutral transport UCPs are proton transporters The intermediate used to convert redox energy into ATP is the proton electrochemical gradient AMHT When a higher turnover of redox equivalents is needed shown here as NADH turnover the increased proton pumping rate increases the electrochemical potential of the membrane which in turn makes proton pumping more difficult unless ATP turnover is increased To increase the flexibility of the system and allow re oxidation without changing ATP turnover two mechanisms can operate One possibility is to change the stoichiometry of proton pumping through the respiratory chain or the stoichiometry of proton utilization by ATPase Such a phenomenon is known as slipping of the respiratory chain The second type of mechanism referred to as respiration uncoupling is the dissipation of the proton gradient across the inner membrane through UCPs which catalyse proton leaks not coupled to ATP production When the membrane is leaky to protons proton pumping by the respiratory chain occurs at a higher rate of respiration without changing the ATP production level or AMHT Respiration uncoupling facilitates NADH reoxidation without increasing ATP production When respiration is uncoupled the increased oxidation rate generates heat as a by product We propose that this pathway was originally present for metabolic purpose and was transformed into a thermogenic pathway in the case of the UCPi in the BAT of mammals In this respect proteins such as UCP2 or UCP3 may represent the ancestors of UCPT expressed in skeletal muscle and although the expression of UCP2 is broad there are probably cells that do not express it such as hepatocytes in normal rats 70 Therefore UCP2 and UCP3 are not absolutely required for normal mitochondrial function On the other hand it remains possible that UCP2 and or UCP3 are UCPs that are subject to speci c regulation operating in several cell types When the mitochondrial membrane potential is close to 180 mV then assuming that the mitochondrial inner membrane is 10 nm thick the resulting electrical eld is greater than 105 V cm With such an electrical eld the occurrence of leaks across a membrane able to perform many ionic exchanges is not surprising On the other hand the observation that the leak could vary according to the physiological situation suggested that the leak was something more than an unavoidable waste of energy By maintaining respiration active the leak helps re oxidation of coenzymes which is associated with heat production and an increase in energy expenditure Several explanations for this leak and the associated waste of energy have been put forward the need for thermogenesis the control of metabolic uxes and the maintenance of a correct redox balance including defence against reduced forms of coenzyme Q which constitutes a source of hazardous free radicals reviewed in 2028 The discovery of new genes closely related to that of the BAT UCP which catalyses the regulated proton leak led to the proposal that UCP2 and UCP3 participate in regulation of the proton leak Different experimental approaches and in particular reconstitution studies in liposomes 112 support a proton translocating activity of UCP2 and UCP3 Analysis of tomato mitochondria also revealed a protontranslocating activity of 2000 Biochemical Society plant mitochondria 117 Table 2 summarizes data for and against an uncoupling activity of UCP2 UCP3 and StUCP Uncoupling of respiration may regulate the level of coenzymes and metabolic pathways Lipogenesis from acetylCoA consumes ATP and requires NADPH but the rst steps of lipogenesis from glucose generate NADH which when oxidized by mitochondria results in ATP formation Flatt 118 calculated that surprisingly the synthesis of fatty acids from glucose may result in a net synthesis of ATP Since a signi cant part of this ATP comes from mitochondria re oxidizing NADH the coupling state of mitochondria could limit the speed of fatty acid synthesis from glucose Therefore the rate at which an animal could store energy would be limited by the capacity of adipocytes to waste it 29 In fact this is exactly what happens in brown adipocytes which have to maintain con comitant lipogenesis and fatty acid oxidation In these cells uncoupling of respiration through UCPl limits ATP synthesis activates the respiratory chain and maintains oxidation of NADH 29 Although no data are available it may be hypothesized that the roles of UCP2 and UCP3 could be to maintain the mito chondrial membrane potential below a certain value limit ATP production by the mitochondria otherwise in the presence of high levels of substrates an increase of ATP would inhibit the respiratory chain maintain the oxidation of reduced coenzymes control the ratio between oxidized and reduced coenzymes and modulate lipogenesis ketogenesis and amino acid metabolism This proposed role is depicted in Figure 4 Uncoupling proteins 169 raUCP1 rUCP1 mUCP1 haUCP1 hUCP1 pUCPZ rUCPZ mUCPZ hUCPZ rUCP3 E mum hUCP3 I UCPArabidopsis UCPSohnum rMDCC rbOGCP hOGCP rOGCP hUCP4 1 F 39mBMCP1 hBMCP1 rGDC L cMPCP rMPCP rADT1 hADT1 hADT3 hADTZ rADTZ Figure 5 Evolutionary tree of UCPs and other mitochondrial carriers The length of horizontal branches is proportional to distances between sequences Abbreviations MDCC mitochondrial dicarboxylate carrier OGCP oxoglutarate malate carrier BMCP brain mitochondrial carrier protein MPCP mitochondrial phosphate carrier protein GDC Graves disease carrier protein ADT adenine nucleotide translocator or ADPATP carrier ra rabbit m mouse r rat ha hamster h human p pig b bovine The plant UCPs Arabdopss halama UCP and Solanum tuberosum UCP are more closely related to mammalian UCPs than to other mammalian mitochondrial carriers UCP4 refers to a gene expressed in brain and encoding a putative protein moderately similar to UCP1 UCP2 and UCP3 All sequences are available from GenBank Regulation of reactive oxygen species ROS During respiration a small proportion of the oxygen molecules are converted into superoxide radicals 02quot by the oneelectron reduction of 0 mainly by mitochondrial complexes I and III 119120 HZO2 is produced as a secondary product via OZquot dismutation Moreover HZO2 itself is the source of more reactive intermediates such as the hydroxyl radical OH Under hyper oxic conditions mitochondrial OZquot generation is increased linearly with oxygen concentration 120 ROS are also produced in the destruction of cells during infection in the degradation of fatty acids and other molecules by peroxisomes and as by products of processes that defend against toxic chemicals 121 Excess superoxide ions may cause peroxidation of phospholipids damage mitochondrial DNA alter proteins or induce trans criptional factors 122 Oxygen radicals can damage DNA phospholipids and proteins and also affect the transcription of genes The State 4 conditions of high redox pressure combined with a limited rate of utilization of the H gradient in an oxygenrich environment promotes OZquot formation and oxygen radical production decreases when ADP phosphorylation increases State 3 123 Skulachev 28 proposed that aerobic cells develop a special protective system against toxicity and suggested the existence of the mild uncoupling of respiration and phos phorylation by means of increased H leak through the mito chondrial inner membrane In fact uncouplers increase the rate of electron transfer and inhibit OZquot formation by mitochondria 119 The explanation of this effect is that partial uncoupling stimulates O2 consumption shortens the lifetime of CoQquot which is an excellent oneelectron O2 reductant and an initiator of ROS formation and inhibits ROS production 123 Therefore respiration uncoupling represents a powerful system for the limitation of ROS production This suggested that one of the functions of proteins that uncouple respiration could be the limitation of ROS synthesis Interestingly NegreSalvayre et al 124 have shown that this system operates in BAT mitochondria These authors observed that HZO2 production was stimulated by the inhibition of BAT mitochondrial respiration through the addition of 04 mM GDP which directly inhibits UCP1 The same authors also observed a similar effect of GDP on mito chondria isolated from the spleen or thymus Since these tissues contain no UCP1 but express the UCP2 gene the authors concluded that UCP2 is a regulator of mitochondrial HZO2 production 124 Although such a role for UCP2 may exist the conclusion of the authors remains questionable since the in hibition of UCP2 activity by GDP was not demonstrated and since no inhibition or a partial inhibition of UCP2 by nucleotides has been observed by others 112115 Diehl and her collaborators have recently obtained data supporting a role for UCP2 in the response to mitochondrial oxidant production in macrophages and hepatocytes of obob mice They reported changes in UCP2 mRNA and protein levels associated with HZO2 production 72125 These authors tested the hypothesis that tumour necrosis factora increases mito chondrial oxidant production after partial hepatectomy They observed that the tumour necrosis factorocdependent increase in oxidant production by liver mitochondria promoted UCP2 induction which may represent an antioxidant defence mech anism 122 The same authors observed an increased level of UCP2 in hepatocytes cultured in the presence of lipid emulsion 126 In that study lipids increased ROS formation prior to UCP2 induction in hepatocytes It was proposed that the liver may adapt to an excessive supply of substrates by inducing UCP2 to facilitate substrate disposal see Figure 4 while containing ROS production 126 A role for UCP2 in fatty liver to protect hepatocytes against apoptosis was also proposed 127 Evolutionary aspects UCPs belong to the superfamily of anion carriers of the mito chondrial inner membrane The characterization of an un coupling mechanism in a protozoan strongly suggests that the UCPs emerged as specialized proteins for proton transport early during phylogenesis and occur in the whole eukaryotic world 62 Figure 5 shows that the three animal UCPs and the plant UCPs have the same root The closest related mitochondrial carriers are the mitochondrial dicarboxylate carrier and the oxoglutarate carrier whereas the genes for the mitochondrial phosphate carrier and ADP ATP carrier are slightly more distant in evolutionary terms It is therefore tempting to speculate that the proton transport of UCP1 originates from a previous activity related to an energyconserving process It has been suggested that UCP1 was derived from an anionproton symporter 39 The membrane potential of respiring mitochondria opposes the net accumulation of anions in this organelle and therefore 2000 Biochemical Society 170 D Ricquier and F Bouillaud proton cotransport would ensure electroneutrality as with the phosphate carrier 97 The binding of a nucleotide to UCPl induces a conformational change that results in the marked stability of the interaction of the nucleotide with UCPl and is consistent with the hypothesis of an aborted transport of nucleotide This suggests that nucleotides could have been transported by an ancestor of UCPl Therefore a possibility is that UCPl originated from a protonnucleotide symporter in which conformational changes induced by nucleotides regulate the proton pathway A further proposal is that the binding of fatty acids could open a bypass allowing protons to go through independently of the process of normal transport A parallel has been drawn between UCPl where fatty acids could act as prosthetic groups and bacteriorhodopsin where the prosthetic group is the retinal bound to the protein 128 Moreover it was noticed that in certain conditions the bacterio rhodopsin molecule can transport either proton or chloride 129 This characteristic was also observed for UCPl To summarize we propose two working hypotheses i UCPl activity changes according to the absence or presence of fatty acids and also according to the amount of fatty acid and ii the activities of UCP2 and UCP3 are more related to the activity of UCPl ancestors than to that of UCPl and the physiological relevance of their transport activity and or uncoupling activity is unknown GENETICS 0F UCPs The genomic organization of the mouse rat and human UCPI genes has been analysed in several laboratories 78130 145 The structure of the U CPI gene is highly conserved in these three species The UCPI gene is composed of six exons encompassing the coding sequence and every exon encodes a membranous domain of the protein Figure 6 The structure of the U CP2 and l1kb UCP1 ATG 39 TGA 1 Iil 1U C C l I i Exons I II IN V VI UCP2 ponAsite UCP3 1 ATGS ATG TGA 391 in I I n Exons I II III VIVII IV VI VIII UCP3 1 V ATG TGAs TGAL H SE US Exonsl II IN V I VII Figure 6 Alignments oi the UOP1 UCP2 and UOP3 genes Exons are indicated using roman numerals Translation initiation ATG emboldened and termination TGA codons are shown The 1 position of the UCP2 gene is located 70 or 82 kb respectively downstream of the polyA site of the human or mouse UCPS gene 149 Human UCP3 mRNAs exist in short TGAS and long TGAL forms Untranslated exons of UCP2 and UCPS genes are shown as black boxes Translated codons are shown as pink boxes 2000 Biochemical Society UCP3 genes is similar to that of the UCPI gene the coding sequence being distributed over six exons in each of the three genes However the UCP2 and UCP3 genes differ from the UCPI gene by the presence of two or one untranslated exons located on the 5 side of the UCP2 and U CP3 gene respectively 146 151 The structure of the genes of plant UCPs is somewhat different since nine exons covering the coding sequence were detected in the gene encoding AtUCP M Laloi unpublished work The mouse rat and human U CPI genes have been assigned to chromosomes 8 130 19 152 154 and 4 133 respectively Following the initial cloning of UCP2 and UCP3 cDNAs it was observed that these two genes are within less than 100 kb of each other 57146155 In fact it was demonstrated that the two genes are adjacent the UCP2 gene being only 7 or 8 kb downstream of the U CP3 gene in mouse and human respectively 149151156 The immediate vicinity of the two genes strongly suggested a duplication event The U CP3 U CPZ locus is localized on mouse chromosome 7 human chromosome 11 3057146 and rat chromosome 1 155 Functional dissection of the UCP1 gene Detailed studies yielded rather good knowledge of the molecular mechanisms regulating U CPI gene transcription for reviews see 78 The two areas to be studied were i the mechanisms explaining the unique expression of UCPl in brown adipocytes and ii the identi cation of genomic regions and transcriptional factors participating in the regulation of transcription in response to physiological situations such as exposure of rodents to the cold In fact it was shown that the same genomic region controls both the tissuespeci c expression of the gene and its response to hormonal factors Studies in transgenic mice demonstrated the essential role of the 5 end of the mouse 157 or rat UCPI gene in its speci c expression in BAT 134141158 CassardDoulcier et al 134 identi ed a potent 200bp enhancer located at position 24 kb in the rat gene This enhancer was also identi ed in the mouse UCPI gene 135 as well as in the human UCPI gene M M GonzalezBarroso D Ricquier and A M Cassard Doulcier unpublished work The UCPI gene enhancer has a complex organization and mediates the transcriptional activation of the UCPI gene by retinoids thyroid hormone cAMP activator protein1 AP1 related factors and thiazolidinediones which are synthetic counterparts of fatty acids and certain prostaglandins The response elements for these factors were functionally identi ed in the enhancer 132136 141159 Response elements for retinoids seem to be particularly important for the control of U CPI gene transcription This is in agreement with previous studies dem onstrating that thiazolidinediones were able to induce UCPI gene expression not only in brown adipocytes 160 but also in nondifferentiated 10T1 2 broblasts 161 or in rabbit cervical preadipocytes which do not express UCPl under basal con ditions 162 It was recently demonstrated that the 200bp enhancer alone not only mediates hormonal responses but is su icient to drive the speci c expression of a reporter gene to BAT 158 A new coactivator referred to as PGCl peroxisome gamma coactivator 1 was identi ed in BAT and skeletal muscle this protein coactivates the transcriptional effect of peroxisome proliferatoractivated receptor 7 and thyroidhormone receptors in the U CPI gene 142 Other elements present in the proximal region of the UCPI gene promoter also mediate regulation of UCPI gene transcription to cAMP C EBP CCAATenhancer binding protein ac CEBP and cJun 135143 145 Uncoupiing proteins 171 Table 3 Linkage or association studies oi the human UOP1 UCPZ and UCPZ genes RMR resting metabolic rate BMi body mass index ratio between height m and square 0t bodyweight kg2 The genetic markers used were either DttSQtt and DttSQtB which are anonymous markers close to the tCPZtCPS locus or the indicated The exon 6yariant corresponds to a the WW gene N 8 iiOiirSigiiiliCaiil statistical difterence Reterences are given in the text variants The 45 bp exon 8 yariant corresponds to a mutation oi the human tCPS gene at the level oi the splice donor site 0t exon 6 resulting in shortening ot the protein lyrrggrlyr is a silent mutation in 45pr sequence which was present or absent in exon 8 ot the human tCPZ gene UCP7 UCPZ UCPS Trait Pyaiue Marker Trait P value Marker Trait P value Marker MR P 0 000002 Diisgii P0006 DiiSQiB Anorexia nervosa P00002 Diisgii P 0 09 Di 1 916 Obesity P NS DiiSQiB Fat gain Plt 0005 3738268 Sleeping energy expenditure Decreased tat oxidation Exon 6 splice P00i6 Aia755gtVal P0019 P00t6 45bp exon8 Weight gain Plt 0002 3738268 BMi P 0 0t8 45 bp exon 8 Obesity P 002 lyrrggrlyr Leptinaemia P 0006 45 bp exon 8 Juvenile obesity P N S lyrrggrlyr Weight loss Obesity Weight loss atter diet Plt 0005 3738268 Weight loss atter 45 bp exon 8 diet P N S BNO P07 Alar55gtVal Functional organization at the UL PZ and UL P3 genes The gross and detailed structure of the human and mouse UCP2 1477150163164 and UCP3 1461511657168 genes have been determined Figure 6 A particular feature of the mouse and human UCP2 genes is the presence of several ATGs inframe with an open reading frame for an unknown peptide of 36 amino acids in exon 2 whereas the UCP2 coding sequence begins in exon 3 Exon 2 did not prevent UCP2 synthesis in vitra and it was demonstrated that the UCP2 initiation codon for translation is in exon 3 149 cDNA cloning of UCP3 revealed that the human gene is expressed as two splice variants generated by alternative splicing of the last intron the predicted amino acid sequences correspond to a putative 312aminoacid protein UCP3L and a 275aminoacid protein UCP3S lacking the last potential transmembrane domain 5556 The two UCP3 mRNAs are expressed in similar amounts in human skeletal muscle 146169 The existence of two forms of UCP3 mRNA has not been reported in rodents Unlike the UCPI gene the mouse and human UCP2 promoter regions lack TATA and CAAT boxes but are GCrich 148149 Primer extension experiments have identi ed transcription start sites in the mouse and human UCP2 genes 148149 Possible regulatory elements for Spl AP2 APl CREB cAMPresponse elementbinding protein MyoD glucocorticoid receptor and peroxisomeproliferatoractivated recep ors e 148149170 Sequencing of DNA upstream o the tran scriptional start site of the mouse and human UCP2 genes also revealed the presence of a consensus site for nuclear factor KB C Pecqueur S Raimbault and D Ricquier unpublished work Although the functional activity of these binding sites for various factors has not yet been demonstrated a possible role for these sites was suggested by the observed effects of thiazolidinediones phorbol esters and endotoxin on UCP2 mRNA levels in animals or in cultures 1221717175 Yamada et al 148 reported that 1250 bp of the promoter region of the mouse UCP2 gene showed signi cant promoter activity in L6 3T3 L1 HeLa NIH 3T3 and CVl cells in agreement with the ubiquitous expression of the gene Interestingly these authors measured a very high promoter activity in GHACl cells derived from rat pituitary The same authors observed that the region between bp 7160 and 7678 exhibited a strong positive regulatory activity A signi cant activity of 1400 bp of the promoter region of the human U gene was measured in 1B8 adipocytes 149 It was also shown that this region contains an activatory element 149 Another study revealed that the mouse UCP2 promoter responded to the cAMPdependent protein kinase and delineated an enhancer element between bp 7233 and 734 170 The same authors identi ed silencer elements upstream of position 7602 and in intron 1 170 Others reported that the coactivator PGCl controls mitochondriogenesis and was able to induce UCP2 mRNA in myocytes 176 The 5 anking region of the human UCP3 gene has been deposited in GenBank accession no AF032871 and was also described in two papers reporting the presence of a putative TATA box in both the mouse and human UCP3 genes 166168 Acin et al 168 identi ed the transcriptional start site of the human UCP3 gene and described several potential binding sites for regulatory factors and motifs for the Ebox MyoD myocyte enhancer factor2 peroxisomeproliferatoractivated receptors and thyroid hormones The presence of such recognition motifs is in agreement with the expression of the UCP3 gene in muscles and its regulation by retinoids 177 and 335triiodothyronine 178179 Linkage and association studies at human UCP genes Except for Luft s hypermetabolism syndrome which was de scribed many years before the identi cation of muscle UCPs for review see 121 there is no report of any disease related to the uncoupling of respiration of muscle mitochondria A Bcll polymorphic site located at bp 73826 upstream of the TATA 2000 Biochemical Society 172 D RquUlSl and F Bonillaud box was identi ed in the human UCPI gene and was shown to be associated with fat gain over time 180181 body mass index 182 change in body mass index in response to a hypocaloric diet 183 and the level of UCPl mRNA expression in intra peritoneal fat 184 Several groups reported an additive effect of this UCPI gene variant and the Tip64 4 Tyr mutation ofthe f3 adrenoceptor gene in obese individuals 1857187 In contrast with what was found in other populations no association of the UCPl A3826G sequence variation with obesity was found in Swedish and German cohorts 188189 In other respects several amino acid variants have been identi ed in human UCPl but these genetic variations of the coding region are not common factors contributing to obesity in Caucasians 190191 enetic study of markers encompassing the human UCP2 UCP3 locus revealed a highly signi cant linkage P lt 0000002 with resting metabolic rate 192 Table 3 A linkage to this locus was also observed in patients with anorexia nervosa 193 Kaisaki et al 155 localized the UCP2 and UCP3 genes to a region of rat chromosome 1 linked to glucose intolerance and adiposity in the GotoiKakizaki type II diabetic rat Two frequent polymorphic sites were identi ed in the human UCP2 gene one in exon 4 Ala55 4 Val substitution and another corresponding to a 45bp insertion deletion in the untranslated part of exon 8 1631947197 A rare Ala232 a Thr mutation was also detected in Japanese individuals 198 Walder et al 197 calculated that UCP2 gene polymorphisms were genetically associated with sleeping energy expenditure in Pima Indians whereas no such association was found in a Scandinavian cohort 195 An association between the UCP2 exon 8 variant and body mass index was measured in two groups of South Indians 199 This association was not observed in British subjects but the UCP2 genotype of obese women was correlated with the serum leptin concentration suggesting that the UCP2 variant may affect susceptibility to weight gain by in uencing regulation of leptin 199 No signi cant association between UCP2 poly morphisms and obesity or type II diabetes was noted by others 1471631641941961987200 Several genetic variants have been detected in the human UCP3 gene but no strong association with juvenileonset or adult obesity was calculated 167201202 A polymorphism in the splice donor junction of exon 6 was identi ed by Argyro poulos et al 201 in Gullahspeaking African Americans and in the Mende tribe of Sierra Leone whereas the mutation was not detected in Caucasians Interestingly this polymorphism resulted in loss of the splice junction and premature termination of the protein product which lacked the sixth transmembranous frag ment The authors determined that in individuals bearing this mutation basal fat oxidation was reduced by 50 and suggested a role for UCP3 in metabolic fuel partitioning 201 Table 3 summarizes the data from the genetic studies PHYSIOLOGY 0F UCPs In addition to biochemical and genetic studies physiological aspects of UCPs have been investigated The level of expression of the genes encoding the different UCPs has been measured in various 39quot l quotandl quot sit uations In particular since UCPs are supposed to be implicated in the control of cellular and tissue energetics most physio logical studies have been carried out in animals or humans under conditions affecting energy equilibrium such as exposure to cold or nutritional changes Tables 4 and 5 summarize the effects of various situations hormones or compounds on the expression of the UCPI UCP2 or UCP3 genes In the absence of published 2000 Biochemical Society data referring to null mutants for UCP2 or UCP3 and due to the dif culty of quantifying the proteins with speci c antibodies most studies of expression of the UCP2 or UCP3 genes were carried out through measurement of the mRNA level The putative functions of the UCPs are related to resting or adaptive thermogenesis including fever metabolic adaptation to uxes of substrates energy partitioning and body weight regulation In other respects the high level of expression of the UCP2 gene in macrophages of adult mammals 3070 and in monocytes and macrophages in foetal rodent liver 71 suggest other functions of UCP2 related to in ammation or haematopoietic system development Role ol UCPZ and UCP3 in resting energy expenditure Two independent genetic studies linkage or association studies see Table 3 in Canadians and in PimaIndians 192197 suggested a role for the UCP2 UCP3 locus in determining resting metabolic rate or resting energy expenditure Contrasting data were ob tained during the analysis ofa Swedish cohort 195 In order to approach the biological roles of these UCPs attempts were made to correlate levels of mRNAs encoding the novel UCPs with certain biological parameters Two groups measured a positive association between the UCP3 mRNA level in human muscle 203 or the UCP2 mRNA level in human white fat 204 and resting metabolic rate These studies suggested that the UCP2 and UCP3 genes are determinants of basal energy expenditure in umans Conversely others found no association between the level of UCP2 mRNA or protein in skeletal muscle and resting metabolic rate or resting energy expenditure in obese and diabetic humans 76205 A major role lor UCP1 StUCP and AtUCP in response to cold exposure in animals and plants and a possible role lor UCPZ and UCP3 in lever or control ol temperature ol specitit parts ol the body The role of the regulated uncoupling of respiration of brown adipocytes and of UCPl in adaptive thermogenesis in response to cold exposure was accepted for many years but not fully demonstrated until 1997 The demonstration by Kozak and his collaborators that UCP1 mice were sensitive to cold and unable to maintain their body temperature proved that the function of the UCPI gene is to induce respiratory uncoupling and thermogenesis in order to maintain body temperature in a cold environment 48 Interestingly the authors noted an up regulation ofUCP2 mRNA in the BAT of transgenic mice whose UCPI gene was interrupted Matthias et al 95 found that the level ofUCP2 mRNA in BAT ofUCPl ablated mice was 14fold higher than in BAT of wildtype mice Although it is dif cult to interpret the signi cance of such a regulation of UCP2 it could be viewed as a compensatory mechanism However this com pensation was not ef cient or suf cient since the body tem perature of the coldexposed mice decreased From this it may be inferred that UCP2 cannot signi cantly contribute to thermo genesis in a cold environment In agreement with this we did not nd elevated UCP2 expression in tissues of mice exposed to 4 C for 16 h 30 However other groups reported an increased UCP2 mRNA level in BAT of mice exposed to the cold for 5 h 206 or in BAT heart and skeletal muscle of rats exposed to 5 C for 2 days 207 An upregulation of UCP2 and UCP3 mRNAs was reported in interscapular BAT of rats exposed to 4 C for 7 days 208 Whether these data re ect a true thermo genic potential of UCP2 remains to be demonstrated An increased level of UCP3 mRNA was also measured in BAT of Uncouplrng proterns 173 Table 4 Ettects ot ditterent situations on expression ot UCP genes in rodents and humans Tn the case ot U0 Pt the sarne results were obtarned tor the protern and the mRNA rn the case ot UCPZ or UCPS almost all data correspond to assay ot rnBNA levels VMH Ventrortvtedranr Hypothalarnusrlesroned WAT whrte adrpose trssue 8k rn skeletal muscle Tncreased level 7 decreased level U unchanged Data are trorn reterences quoted rn the text and 2557257 UCPT UCP2 rnBNA UCPS rnBNA Srtuatron BAT BAT WAT 8k rn Lrver BAT WAT 8k rn 0on exposure rodents U U U U U Hrghrlrprd dret rodents Stanatron Bats 7 7 7 Humans Obesrty 017017 rnouse 7 017017 rnouse 7 tata rats 7 7 7 7 VM rats 7 U U Hurna s 7 Drabetes Drabetrc rats 7 7 U 7 Humans wrth type T drabetes 7 Exercrse rats Chronrc 4 Table 5 Ettects ot hormones and other tactors on expression ot UCP genes in rodents Tn the case ot UCPT the sarne results were obtarned tor the protern and the mRNA rn the case ot UCPZ or UCPS all data correspond to assay ot rnBNAs WAT whrte adrpose trssue Skrn skeletal muscle T3 tnrrodothyronrne LBS Trpopolysacchande TNFOL turnour necrosrs tactor OL Leptrn results are those obtarned upon prolonged delrvery ot Teptrn decreased levels ot UCPZ B A and CPS rn N s were rneasured upon acute treatrnentwrth Teptrn 258 reterences quoted rn the text and trorn 2607273 lncreased level 7 decreased level U unchanged Beterences are grven rn the text and Tn 259 Data are trorn UCPT UCPZ rnBNA UCPS rnBNA Compound n W0n W0 BAT BAT WAT 8k rn Lrver BAT WAT 8k rn Fatty acrds n W0 n W0 Thrazolrdrnedrones n W0 7 n W0 Betrnords n W0 n W0 Leptrn n W0 Tnsulrn n W0 3rag t n W0 U U 7 7 7 7 Dexarnethasone n W0 7 7 LBS n W0 n W0 Tnterterony n W0 coldexposed rats by Larkin et al 178 whereas other authors noted no effect of cold on UCP3 gene expression in mice 206 There is agreement that cold exposure does not activate UCP3 mRNA expression in skeletal muscle indicating that this protein is not primarily implicated in body temperature regulation 84178207 The signi cance of high levels of UCP2 mRNA in white fat and of UCP3 mRNA in skeletal muscle of hibernating squirrels is unknown 209 h o enic hormones and factors such as 335triiodo thyronine 571781792107213 leptin 57214215 lipopoly saccharide and tumour necrosis factor0c 173174216217 in crease expression of the UCP2 and UCP3 genes in animals suggesting a contribution to thermogenesis regulated by hor mones or cytokines Induction of UCP2 and UCP3 in tissues of rodents by lipopolysaccharide interleukinlf and other cyto kines and inhibition of the effect of lipopolysaccharide on UCP2 expression y a c clooxygenase inhibitor 173 point to a possible role for the novel UCPs in the still unknown thermogenic mechanisms of fever Although the UCP2 gene is expressed throughout the body it is not expressed uniformly in the organs In the case of the brain UC P2 gene expression is restricted to certain speci c areas such as a few nuclei of the hypothalamus arcuate surprachiamastic and paraventricular nuclei 218 It is tempting to speculate that UCP2 may regulate the temperature and therefore the activity of certain speci c nuclei in the brain The identi cation of UCP homologues in plants 6061 followed reports of the existence of regulated respiration un 2000 Brochernrcal Socrety 174 D RquUlSl and F Bomllaud Table 6 Known and possible biochemical and physiological lunctions ol the UCPs Biochemical actiyity Physiological role UCPt Uncoupling 0t respiration Proton translocation Fatty acid cycling UCPZ and UCPS Uncoupling 0t respiration Proton translocation Fatty acid cycling Uncoupling 0t respiration Unknown mechanism StUCP and AtUCP lhermogenesis and control 0t body temperature Hypothesis control 0t ATP level NADl lNAD r ratio ROS level 7 metabolic adaptation to tatty acid and glucose tluxes Hypothesis resistance to chilling coupling mechanisms in plants 219 Although a true uncoupling activity of plant UCPs has not yet been demonstrated the marked effect of StUCP on mitochondrial membrane potential strongly suggests that this protein is an uncoupler 60 However the exact physiological role of such an uncoupling in plants was unclear Since the UCPI gene is activated upon exposure of animals to the cold the response of the genes of the UCP homologues was tested in plants kept in the cold Surprisingly there was a pronounced increase in StUCP mRNA and AtUCP mRNA levels in Salanum tuberasum and Arabidapsis thaliana respectively when kept at 5 C for 2 days 6061 These data argued for a role for the UCPs in thermogenesis and resistance to chilling in plants They also suggested that uncoupling of respiration can be used to generate heat and that the alternative oxidase pathway is not the only process involved in heat production by plants Whether such a proposal is valid for the different organs of plants remains to be analysed In conclusion the essential role of UCPl in maintaining the body temperature of mice is rmly established Although null mutant mice are needed for de nitive conclusions to be drawn the UCP2 and UCP3 genes do not seem to be related to adaptation to the cold although a role in regulation of body temperature or the temperature of particular parts of the body or a role in fever cannot be excluded In other respects increased expression of the UCP2 and UCP3 genes in skeletal muscle during starvation or immediately after the cessation of exercise see below is inconsistent with a role for these UCPs in thermogenesis and wasting of energy Conversely the marked upregulation of plant UCP mRNAs in organs of plants kept at 4 C suggests a thermogenic role for the plant UCPs or at least a role in defence against the cold Contribution ol UCPs to DIT energy partitioning and lipid metabolism Utilization of food induces extra heat production This corres ponds to obligatory and adaptive energy dissipation due to digestion and metabolism and is referred to as DlT Previous studies have shown that BAT can buffer excess calorie intake under certain nutritional conditions in rats 34 Theoretically any increase in respiratory uncoupling in response to food intake induces thermogenesis and decreases food ef ciency Therefore the UCPs may be considered as candidates for the regulation of DlT and energy partitioning The role of UCPl in the wastage of energy generated by diet was demonstrated in starved animals 9220 and in transgenic mice overexpressing UCPl in their white adipose tissue 221 or underexpressing UCPl in their BAT 222223 2000 Biochemical Society UCP2 and UCP3 also appear to be able to control energy partitioning and food ef ciency A highlipid diet activated UCP2 expression in white fat of mice and in particular in two strains of mice which are resistant to dietinduced obesity 3058156 In addition a twofold induction of the UCP3 mRNA level in skeletal muscle of rats or mice receiving a high fat diet was also reported 58151 Compared with carbohydrate feeding shoil feeding upregulated UCP2 mRNA in liver and UCP3 mRNA in skeletal muscle ofmice 224 Samec et al 225 measured a marked increased in UCP2 and UCP3 mRNA levels in the gastrocnemius muscle of rats put on a restricted diet and then fed on a highfat diet During the analysis of UCP2 and UCP3 mRNA levels in obese individuals after stable weight reduction or during prolonged weight loss expression of the UCP3 mRNA level decreased suggesting that reduced UCP3 expression could contribute to decreased energy expenditure in weightstable weightreduced individuals 226227 A role for fatty acids in upregulating UCP3 expression in skeletal muscle was con rmed by infusing rats with a lipid emulsion and heparin 228 In vitra and in viva experiments demonstrated that fatty acids or certain retinoids promote UCP2 and or UCP3 expression in white and brown adipocytes myo cytes and pancreatic islets 1 71 172 1772062297238 Expression of UCP2 andor UCP3 in skeletal muscles heart or adipose tissue can be induced by various situations characterized by elevated levels of circulating nonesteri ed fatty acids such as starvation in humans or rodents 84169239240 highfat feed ing in rodents 30177 and genetic or experimental diabetes in rodents 31241242 It is not known whether the very high level of ketone bodies observed in diabetic rats is related to UCP induction 241 The increased expression of the UCP2 and UCP3 genes in skeletal muscle of starved rats 207 or humans 239 questions their exact roles and argues against a thermogenic role for UCPs suggesting instead regulation of lipids or lipid metabolites as substrates 84240243 In terms of this hypothesis although the precise roles of UCPs in lipid metabolism remain to be elucidated it should be recalled that a possible role for UCPs is to transport fatty acids into or out of mitochondria see section on biochemistry of UCPs Such a hypothesis is in reement with studies of UCP2 and UCP3 reconstitution in liposomes 112 UCPs and glucose utilization a role in insulin resistance The observed increase in expression of muscle and heart UCP3 or UCP2 mRNA in rodents with type 1 diabetes 241242 and in humans with type II diabetes 20 5 is not easy to interpret since several metabolic parameters are altered in diabetes However Uncoupling proteins 175 recent tiidie 39 rquot CPS in 39 insulin resistance and eveninsulin secretion Matsuda et al 58 proposed that increased expression of UCPs may provide a defence against highfatinduced obesity and impairment of glucose metabolism Interestingly there is a correlation between UCP3 mRNA levels in the human vastus lateralis and wholebody insulinstimulated glucose uptake 75 Similarly Western analysis of the role of UCP2 and UCP3 in the partitioning of metabolic fuels in humans suggested that muscle UCP2 and UCP3 levels were correlated with carbohydrate oxidation and higher insulin mediated glucose uptake 244 Other data supporting a link between muscle UCPs and glucose utilization have come from studies of exercised or immobilized animals Increased expression of UCP3 and UCP2 mRNA levels was observed in muscles of exercised mice 073 h after the end of the last bout of exercise under conditions where GLUT4 levels and insulininduced glucose uptake are increased 245246 Conversely 20730 h after cessation of the last bout of exercise when the insulininduced glucose uptake has returned to its basal value no increase in UCP3 or UCP2 mRNA was observed 245247 In addition an upregulation of skeletal muscle UCP3 mRNA was measured in immobilized rats under conditions where glucose uptake was increased M Marzolo D Ricquier X Bigard and B Serrurier unpublished work Studies based on the use of antidiabetic compounds such as thiazolid inediones also support a role for muscle UCPs in glucose utilization 172230248 Interestingly Hjeltnes et al 249 mea sured an increased level of UCP2 and UCP3 mRNAs in skeletal muscle from tetraplegic subjects and a normalization after electrically stimulated leg cycling under conditions where insulin stimulated glucose uptake was increased Shimokawa et al 248 demonstrated that the UCP3 mRNA level in skeletal muscle was correlated strongly with levels of circulating glucose in rats treated with a thiazolidinedione suggesting that the thiazol idinediones increased glucose catabolism by upregulating UCP2 expression Samec et al 250 reported a positive association between muscle UCP2 and UCP3 mRNA expression and increased plasma glucoselevels over 2 h after a glucose load in rats They concluded that the upregulation of muscle UCP2 or UCP3 by a highfat diet may be more closely linked to insulin resistance than to changes in circulating nonesteri ed fatty acids These authors proposed that such a positive association between muscle UCP2 or UCP3 expression and insulin resistance would be consistent with a role for muscle UCPs in the regulation of lipid as a substrate because in the early stages of the development of insulin resistance increased fatty acid oxidation limits glucose utilization by muscle cells in agreement with the Randle hy pothesis 251 TsuboyamaKasaoka et al 252 measured an up regulation of UCP3 mRNA in skeletal muscles of transgenic mice with increased GLUT4 content they suggested that in creasing the rate of glucose uptake was inducing UCP3 expression and that UCP3 was involved in glucose utilization by skeletal muscle The UCP2 gene is expressed at high levels in pancreatic islets 30229253 Upregulation of UCP2 associated with increased fatty acid oxidation in pancreatic islets of rats receiving leptin has been described 253 Since it is known that the ATPADP ratio in cells controls insulin secretion and that ATP par ticipates in granule priming and exocytosis it is tempting to speculate that UCP2 could modulate the secretion of insulin through changes in ATP Wang et al 73 reported that over expression of UCP2 in pancreatic islets of Zucker diabetic fatty rats normalized 39 f l 39 insulin secretion whereas Chan et al 74 decribed an inhibition of 39 quot quot quot frmratquott 39 UCP2 This latter report agrees with an uncoupling activity of this protein a decreased ATP content and the expected reduced insulin secretion It supports a potential role for UCP2 as an important modulator of cell function 74 CONCLUSIONS AND PERSPECTIVES Animal and plant UCP homologues form a subfamily of mito chondrial carriers that are evolutionarily related and possibly derived from a protonanion transporter The BAT UCPl induces uncoupling of respiration and thermogenesis to maintain body temperature at 37 C The plant UCPs may be concerned with response to the cold and resistance to chilling The bio chemical activities and biological roles of the recently identi ed UCP2 and UCP3 are poorly understood see Table 6 Analysis of recent data points to a role for these UCPs in fatty acid or glucose utilization regulation of ATP production by mito chondria control of the NADHNAD r ratio and limitation of the level of ROS Whether these UCPs contribute to proton leaks measured in mitochondria from most tissues remains to be demonstrated in viva Data from genetic studies and mea surements made in humans support a contribution of UCP2 and U P3 to resting energy expenditure Major goals for future research will be the identi cation of the structure of these novel UCPs as well as the analysis ofmice null for the UCP2 or UCP3 gene Other aims will be to investigate the roles of UCP2 and UCP3 in insulin secretion control of temperature in brain nuclei ammatory processes fever response to oxidative stress cell t 1n proliferation and apop os1s We express our gratitude to Serge Raimbault and Dr Daniel Sanchis for help in preparing the Figures and to Dr DaVid Marsh for critical reading of the manuscript D R and E B are established CNRS and lNSERM investigators respectively Our research is supported by Centre National de la Recherche Scientitigue CNRS Human Frontier Science Program organization RESP Association de Recherches sur le Cancer ARC Association Erancaise contre les Myopathies AEM Human Training Mobility Programme of the European UlllOll and lnstitut de Recherches lnternationales SerVier REFERENCES 1 NlCROllS D G and Locke R M 1984 Physiol Rev 64 1764 2 Cannon B and Nedergaard J 1985 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