Activating {omega}-6 Polyunsaturated Fatty Acids and Inhibitory Purine Nucleotides Are High Affinity Ligands for Novel Mitochondrial Uncoupling Proteins UCP2 and UCP3*

Markéta Zácková, Eva Skobisová, Eva Urbánková and Petr Jezek {ddagger}

From the Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 14220 Prague 4, Czech Republic

Received for publication, December 17, 2002 , and in revised form, March 31, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human UCP2 and UCP3, expressed in yeast, were studied to establish their high affinity regulatory ligands. UCPn were reconstituted into liposomes and assayed for fatty acid (FA)-induced H+ efflux. All natural long chain FAs activated UCP2- and UCP3-mediated H+ translocation. Coenzyme Q10 had no further significant activating effect. Evaluated parameters of FA activation (FA cycling) kinetics revealed the highest apparent affinity to UCP2 (the lowest Km values: 20 and 29 µM, respectively) for {omega}-6 polyunsaturated FAs (PUFAs), all-cis-8,11,14-eicosatrienoic and all-cis-6,9,12-octadecatrienoic acids, which are also the most potent agonists of the nuclear PPAR{beta} receptor in the activation of UCP2 transcription. {omega}-3 PUFA, cis-5,8,11,14,17-eicosapentaenoic acid had lower affinity (Km, 50 µM), although as an {omega}-6 PUFA, arachidonic acid exhibited the same low affinity as lauric acid (Km, ~200 µM). These findings suggest a possible dual role of some PUFAs in activating both UCPn expression and uncoupling activity. UCP2 (UCP3)-dependent H+ translocation activated by all tested FAs was inhibited by purine nucleotides with apparent affinity to UCP2 (reciprocal Ki) decreasing in order: ADP > ATP ~ GTP > GDP >> AMP. Also [3H]GTP ([3H]ATP) binding to isolated Escherichia coli (Kd, ~5 µM) or yeast-expressed UCP2 (Kd, ~1.5 µM) or UCP3 exhibited high affinity, similar to UCP1. The estimated number of [3H]GTP high affinity (Kd, <0.4 µM) binding sites was (in pmol/mg of protein) 182 in lung mitochondria, 74 in kidney, 28 in skeletal muscle, and ~20 in liver mitochondria. We conclude that purine nucleotides must be the physiological inhibitors of UCPn-mediated uncoupling in vivo.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Novel mitochondrial uncoupling proteins, ubiquitous UCP2,1 predominantly muscle-specific UCP3 and brain-specific UCP4, and BMCP (or UCP5) form a gene subfamily within the mitochondrial anion carrier gene family together with "classic," brown adipose tissue-specific UCP1 and at least three distinct plant UCPs (1, 2). Three basic physiological roles of all UCPs should be manifested as consequences of low uncoupling states (2): (i) a slight acceleration of metabolism caused by slightly increased respiration, (ii) a concomitant reduction in reactive oxygen species (ROS) formation (3, 4), and (iii) inevitably related, mild thermogenesis. In addition, UCP2 (UCP3) most probably possesses more specific physiological roles (2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) in defense against excessive ROS production (2- 5), in participation in body weight regulation (5, 6, 7, 8, 13); in heat production during various types of adaptive thermogenesis (8, 9, 14), including fever (9), and in switching between pro- and anti-apoptotic processes (15). Pathologically distorted regulations of UCP2 (UCP3) expression or distorted ligand regulations are most probably among the causes of the development of obesity (13), type 2 diabetes (16), heart failure (5), and aging.

The diverse UCPn functions require fine-tuned regulations that can be accomplished only by regulation and up-regulation of their transcription (e.g. see Refs. 5, 8, 9, 14, 17, 18) and by down-regulation of their translation (19). Biochemical regulation, i.e. regulation by ligands, cofactors, or by covalent modification are also likely involved. The first line of regulation could be provided by FAs (20). It has been established that, like UCP1 and plant UCP (PUMP), UCP2 and UCP3 are also activated by FAs (20, 21), most probably because of the FA cycling mechanism (20, 22, 23, 24). Hence, the availability of free FAs is the first prerequisite for UCP function. Second, purine nucleotides (PN) were confirmed to inhibit the protonophoric action of reconstituted Escherichia coli-expressed UCP2 or UCP3. Garlid and co-workers (20) observed a lower PN affinity toward UCP2 and UCP3 in sulfate media (20), whereas Klingenberg and co-workers (21) reported a higher affinity in low salt medium. Modulation, i.e. the lowering of PN inhibition by some yet unknown factors, may represent the mechanism that activates UCP2- or UCP3-mediated uncoupling in vivo. Such well known modulators for UCP1 are alkaline pH and Mg2+ (24, 25). Also, cofactors like coenzyme Q10 (21, 26) and superoxide (27) were suggested to activate UCP2 (UCP3). Coenzyme Q10 was claimed to be required to restore the intact conformation of recombinant UCPs solubilized by sarcosinate from inclusion bodies (21, 26). Even native UCP2 (UCP3) was reported to be stimulated upon superoxide generation in various types of mitochondria (27). UCP2 (UCP3) in this presumably stimulated state was inhibited by ATP, ADP, GTP, and GDP in kidney or skeletal muscle mitochondria (27). These results suggested that native UCP2 (UCP3) could also be inhibited by PN. Recently, fluorescent nucleotide derivatives were demonstrated to interact with E. coli-expressed UCP2 (28), but direct measurements with native nucleotides were still lacking.

Most initial studies of recombinant UCP2 and UCP3 expressed in yeast were performed while monitoring coupling in yeast mitochondria (6, 7, 8). However, available reports contain numerous contradictory results (29, 30, 31, 32, 33, 34, 35, 36). Claims were made that UCP2 is specifically activated (state 4 respiration in yeast mitochondria is stimulated) by all-trans-retinoic acid and that it is insensitive to palmitic acid, 9-cis-retinoic acid, and other FAs tested including arachidonic, linoleic, docosahexaenoic acid, and prostaglandin E2 (36). It is difficult to imagine a physicochemical basis for this strange FA specificity, which is incompatible with previous surveys of FAs interacting with UCP1 (37, 38).

Hence, in this work we have studied reconstituted yeast-expressed UCP2 and UCP3 in their partially purified (but highly active) form, in order to screen in detail their natural ligands, FAs and PN. We have clearly demonstrated that all natural long chain FAs, saturated or unsaturated, activate UCP2- or UCP3-mediated H+ translocation. From kinetics of putative FA cycling we have found the highest apparent affinity to UCP2 (lowest Km) for two {omega}-6 polyunsaturated FAs, which are also the most potent agonists of PPAR{beta} (17). This finding suggests their possible dual role in activating both UCP expression and uncoupling activity. We have also shown that the apparent affinity to UCP2 (reciprocal Ki) is decreasing in order: ADP > ATP ~ GTP > GDP >> AMP. Demonstrating the existence of high affinity [3H]GTP and [3H]ATP binding to recombinant UCP2 (Kd, ~1.5 µM for yeast expression) and UCP3, expressed either in yeast or E. coli (Kd, ~ 5 µM) and showing that numbers of very high affinity [3H]GTP binding sites (Kd, ~0.2–0.4 µM) decrease in the mitochondria of lung > kidney > muscle > liver, we provide strong support for the view that PN must be the physiological inhibitors of UCPn-mediated uncoupling in vivo.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Most of the chemicals were purchased from Sigma. 3H-labeled nucleotides were from Amersham Biosciences. Hydroxylapatite, Bio-Gel HTP, and Bio-Beads SM2 were from Bio-Rad. Octylpentaoxyethylene (OctylPOE) was from Bachem Feinchemikalien, Bubendorf, Switzerland. Materials for reconstitution were from the same sources as described elsewhere (39), materials for yeast fermentation were from Difco. Zymolyase 100T was from ICN. All other chemicals were reagent grade.

Yeast Expression of UCP2 and UCP3—W303 yeast containing pCGS110 (or pYES) vectors with inserted cDNAs coding for human UCP2 and human UCP3 under control of an inducible Gal promoter and ura- selection were donated by Ruth E. Gimeno and Louis A. Tartaglia (Millennium Pharmaceuticals, Inc., Cambridge, MA; Ref. 6). cDNA coding for UCP1 in pCGS110 vector contained in JB516 yeast was from Prof. Karl Freeman (McMaster University, Hamilton, Ontario, Canada; Ref. 40). Yeast cells were grown on ura- selective plates and were inoculated into 4–8 250-ml Erlenmeyer flasks with a medium containing 203 mM (1.5 vol%) lactate, 0.05% glucose, 0.17% yeast nitrogen base (Difco) 0.5% ammonium sulfate, 0.005% each of L-aminoacids (tryptophan, methionine, arginine, leucine, histidine), and adenine sulfate. After 24 h, 0.2% galactose was added, and cells were shaken for another 24–28 h until the optical density of 1 was achieved. Mitochondria were prepared immediately after terminating fermentation using Zymolyase 100T (ICN) to cleave the cell wall (40). Protein extraction followed immediately. Changes in different fermentation yields were compensated by taking the same amount of yeast mitochondrial protein, usually 30 mg.

E. coli Expression of UCP2 and UCP3—Bacterial strains BL21 (Novagen) containing plasmids pET21a with inserted cDNA coding for human UCP2 (or UCP3) open reading frames between the Ndel and Notl sites of the vector (Novagen) were donated by Dr. R. E. Gimeno and Louis A. Tartaglia (Millennium Pharmaceuticals, Inc., Cambridge, MA; Ref. 41). The cells were grown at 30 °C to an OD600 of 0.6 and then induced with 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside at 30 °C for 6 h. Cells from a 700-ml culture were lysed in a French Press in 20 ml of lysis buffer (10 mM Tris, pH 7, 1 mM EDTA, 1 mM dithiothreitol); the lysate was centrifuged at 27,000 x g for 15 min, and the pellet was resuspended in 20 ml of the lysis buffer and centrifuged at 1000 x g for 3 min. 1-ml aliquots of the supernatant were centrifuged at 14,000 x g for 15 min. The resulting pelleted inclusion bodies were stored frozen at -70 °C. When used, they were suspended (3 mg of protein) and washed two times in 10 mM Tris-Cl, 0.1 mM Tris-EDTA, pH 7.0. The washed pellet was solubilized in 0.75 ml of 5 mM TEA-TES, 30 mM TEA2SO4, 0.1 mM Tris-EDTA, pH 7.2, containing 1.67% sodium lauroylsarcosinate (SLS) and 1% octylpentaoxyethylene. The resulted micellar solution was concentrated (thus partly depleted of SLS) and subsequently diluted (usually 1:1) in 20 mM Na-MES, pH 6. Protein content was estimated using the Amido Black method (42).

Isolation and Reconstitution of Yeast-expressed UCP2/UCP3—Reconstitution with lipid protection was adopted from Klingenberg and Winkler (43) to comply with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) fluorescent monitoring of ion fluxes (20, 22, 23, 37, 39). It included OctylPOE extraction of yeast mitochondria (30 mg of protein) under lipid protection, the isolation step on HTP, detergent removal on Bio-Beads SM2 overnight, and washing of external probe Sephadex G25-300. The total amount of added lipid was 40.7 mg (egg yolk lecithin, type XI-E, Sigma, 4% cardiolipin and 1.6% L-{alpha}-phosphatidic acid). Additional lipids in 1 ml of final suspension, up to 20 mg, could have originated from the mitochondria. The internal medium for liposomes contained 84.4 mM TEA2SO4, 28.8 mM TEA-TES with 9.2 mM TEA, pH 7.2, 0.6 mM TEA-EGTA. The protein content of liposomes was also estimated by the Amido Black method (42). Usually, a lipid-to-protein ratio of about 1000 or higher was obtained. The identity of UCP2 (UCP3) in HTP flow-through was verified using peptide mapping assisted by MALDI-TOF mass spectroscopy after in-gel trypsin cleavage of the PAGE-separated acetone-precipitated samples.

Fluorescent Monitoring of H+ Fluxes in Proteoliposomes—Valinomycin-induced H+ fluxes in the presence of various FAs were monitored by the SPQ quenching method (20, 22, 23, 37, 39) with 2 mM SPQ internally. Ethanol solutions of FAs were added to 25 µl(~1.5 mg) of vesicles in 2 ml of external medium (84.4 mM K2SO4, 28.8 mM TEA-TES with 9.2 mM TEA, pH 7.2, and 0.6 mM TEA-EGTA) and H+ efflux was initiated by 0.1 µM valinomycin. Fluorescence was monitored on a RF5301 PC fluorimeter (Shimadzu, Japan), equipped with polarization filters (Polaroid) in cross-orientation in order to decrease light scattering. Fluorescence was calibrated to [H+] by adding KOH aliquots to proteoliposomes in the presence of 1 µM nigericin, and H+ flux rates were calculated as previously described (39). The internal volume was calculated from SPQ volume distribution (20, 22, 23, 39) assuming a total lipid content of 60.7 mg in 1 ml (1.5 mg in the assay) and typically amounted to ~1.2 µl (mg of lipid)-1.

Assay for 3H-labeled Nucleotide Binding—Klingenberg's anion exchange method was used to determine 3H-labeled nucleotide binding (44). Either, E. coli-expressed proteins were used, or the isolation of yeast-expressed UCP2 and UCP3 was modified while the HTP wash fractions were also taken. The samples were further desalted and concentrated using Amicon microconcentrators (cutoff Mr 10,000), to obtain a concentrated micellar protein solution in 10 mM Na-MES, 1.5 mM TEA2SO4, pH 6.5. The optimum protein to nucleotide ratio was adjusted to give linear Scatchard plots (45). Microliter aliquots of [3H]GTP ([3H]ATP) were added to a series of samples (50 µl) or to the parallel sample series with 2.5 mM GTP (ATP, respectively). After 60 min of incubation at 20 °C, the unbound nucleotides were removed by the sample passage through 1-ml spin columns containing Dowex, Cl- form. The flow-through samples were subjected to liquid scintillation counting. The data were calculated using Scatchard plot analysis (45).

Assay for 3H-labeled Nucleotide Binding in Mitochondria—Isolation of mitochondria from rat lung, kidney, skeletal muscle, and liver was performed essentially as described elsewhere (41). Bovine serum albumin-washed mitochondria (0.2 mg of protein) were mixed with [3H]GTP aliquots (8–320 pmol and with 0.6 mM "cold" GTP in parallel series) and incubated for 30 min in 200 µl of 100 mM sucrose, 20 mM Tris-HEPES, 1 mM EDTA, 2 µM rotenone, 5 µM CAT, pH 7.0. Mitochondria were then filtered through nitrocellulose filters (0.45-mm pore, Millipore). The filters were washed twice with sucrose medium and placed into scintillation solution. The measured radioactivity of the samples containing an excess of non-radioactive GTP was always subtracted, and Scatchard plots were generated (45).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
H+ Efflux Induced by Fatty Acids in Proteoliposomes Containing UCP2 and UCP3—Fig. 1, a and b, illustrates the monitoring of H+ fluxes induced by K+ diffusion potential in the presence of lauric acid in proteoliposomes containing the recombinant uncoupling proteins, UCP2 or UCP3. Both were able to mediate H+ efflux upon the addition of 0.1 µM valinomycin to the vesicles pre-equilibrated with 200 µM lauric acid. The lauric acid addition causes the interior acidification of vesicles, called flip-flop acidification, reflecting the redistribution of FA molecules in both leaflets of the lipid bilayer (46). Note that the extent of this acidification is unchanged in the presence of ATP, reflecting the undistorted SPQ response. Consequently, we could detect that the observed H+ fluxes were up to 50% inhibited by various purine nucleotide (PN) di- and triphosphates (vide infra). High PN concentrations also decreased the extent of SPQ response to H+ efflux, which indicates that a single protein per vesicle exists in our preparation. Alternatively, if several proteins exist they have the same orientation. This is feasible because of our high lipid-to-protein ratio,2 and such observations were made for years with UCP1 or plant UCP not only for SPQ monitoring of H+ flux, but also for Cl- and alkylsulfonate flux (22, 23, 37, 40, 47).



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FIG. 1.
ATP sensitivity of lauric acid-induced H+ fluxes in proteoliposomes containing UCP2 (a and d), UCP3 (b), or hydroxylapatite-passed extract from W303 yeast mitochondria (c). Typical traces of intraliposomal H+ (monitored by SPQ) are shown for proteoliposomes equilibrated with 200 µM lauric acid (LA, a–c)or50 µM cis-8,11,14-eicosatrienoic acid (ETA, d). The FA addition leads to so-called flip-flop acidification, resulting from establishing equilibrium with regard to both membrane and acid-base equilibrium (46). A subsequent addition of 0.1 µM valinomycin (Val,1 µM for c) leads to internal alkalization indicating H+ efflux. This H+ efflux was inhibited up to 50% by ATP, but not for the reconstituted HTP-passed detergent extract of W303 yeast mitochondria containing the empty expression vector (c). The right panel in a shows no effect by 1 µM coenzyme Q10 (+CoQ10). Flux densities in 10-6 pmol of H+ s-1 µm-2 were estimated as follows: a, UCP2 control, 62; 1.87 mM ATP, 34; 1 µM coenzyme Q10, 61. b, UCP3 control, 78; 0.75 mM ATP, 40. c, control, 42; 1.87 mM ATP, 42. d, control, 108; 1.87 mM ATP, 48. Control rates expressed in µmol of H+ min-1(mg of protein)-1 were 68 for UCP2 (a) 146 for UCP2 with ETA (d), and 62 for UCP3 (b).

 

Fig. 1 (a and b) illustrates the inhibition of the lauric acid-induced H+ efflux in proteoliposomes containing UCP2 or UCP3. However, ATP did not affect the slow H+ efflux induced at a 10-fold higher valinomycin concentration by lauric acid in proteoliposomes containing the extract from the mitochondria of W303 yeast not expressing UCP2 or UCP3 (Fig. 1c). The W303 extract was prepared and passed through an HTP column similar to that of yeast-expressed UCP2 or UCP3. The resulting FA-induced H+ efflux increased with increasing initial amount of W303 yeast mitochondria. It is likely mediated by yeast carriers of the HTP flow-through, such as the ADP/ATP carrier (48) or the phosphate carrier (39, 49). In turn, the ATP insensitivity of H+ efflux with the reconstituted W303 extract and only slight (<10%) inhibition of FA-induced H+ efflux in UCP2 or UCP3 proteoliposomes by CAT agaric acid,3 or methylenediphosphonate suggest that the ATP-sensitive H+ fluxes in UCP2 (UCP3) proteoliposomes are indeed due to UCP2 (UCP3) function, respectively.

Various natural FAs were able to induce H+ efflux in proteoliposomes containing UCP2 (Table I) and UCP3. Among all tested FAs, the fastest H+ efflux was found for oleic acid and myristic acids followed by polyunsaturated FAs. Also the H+ efflux induced by PUFAs was inhibited up to 50% by the ATP present in the external medium (Fig. 1d and vide infra). Similar results were found with UCP3. Moreover, additions of {omega}-6 PUFAs exhibited the highest extent of flip-flop acidification. Thus, cis-8,11,14-eicosatrienoic acid (C20:3 {omega}-6) had on average a double extent of flip-flop acidification when compared with lauric acid (Fig. 1, a and d). This finding would suggest that the physicochemical properties of FA movement in the membrane and their partition coefficient Kp could contribute to their maximum cycling rates. Also the flip-flop rate of fatty acids was previously found to increase with increasing unsaturation (50). In order to elucidate the involved structure/kinetic relationships we further studied the kinetics of the FA activation of UCP2-mediated H+ efflux.


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TABLE I
Kinetic parameters of putative fatty acid-cycling via mitochondrial UCP2

 



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FIG. 2.
Kinetics of presumed fatty acid cycling mediated by UCP2 lauric acid (a), palmitic acid (b), oleic acid (c), arachidonic acid (d). Left panels show the direct plots of FA-induced H+ flux with increasing total FA concentration for the absence (filled symbols) and presence (open symbols) of 2.5 mM ATP (ADP for a), while the right panels illustrate Eadie-Hofstee plots for the same data. The dotted lines represent theoretical fits by the Michaelis-Menten equation for the differential H+ fluxes, when fluxes in the presence of nucleotides were subtracted from the control ones (Km and Vmax were taken from the linearized Eadie-Hofstee plots). The obtained kinetics parameters are listed in Table I.

 


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FIG. 3.
Kinetics of {omega}-6 eicosatrienoic acid-induced H+ efflux in UCP2 proteoliposomes. cis-8,11,14-Eicosatrienoic acid (ETA)-induced H+ efflux initiated by 0.1 µM valinomycin in UCP2 proteoliposomes was measured for increasing ETA amounts in the absence (•) and presence of 2.5 mM ATP ({circ}). The left panel contains the derived direct kinetic plots, while the right panel shows the corresponding Eadie-Hofstee plot, linearized without taking into account the two highest data points. At these high rates the total H+ flux was influenced by a nonspecific H+ flux (as seen from the diminished ATP inhibitory strength). The dotted lines represent theoretical fits by the Michaelis-Menten equation for the differential H+ fluxes. The obtained Km and Vmax are listed in Table I.

 
Kinetics of UCP2-mediated H+ Efflux Induced by Lauric Acid—Kinetics of lauric acid activation of H+ efflux (FA cycling) in UCP2 proteoliposomes is illustrated in Fig. 2a. H+ efflux rates were evaluated with and without 2.5 mM ADP. Assuming Michaelis-Menten kinetics, we constructed Eadie-Hofstee plots for total rates (right panel in Fig. 2a and Table I) and for differential rates (not shown), when the rates measured with ADP were subtracted from the control rates. The kinetic parameters obtained for differential rates were used to construct the theoretical fit by the Michaelis-Menten equation (dotted line in Fig. 2a). This fit reflects the kinetics of uniformly oriented UCP2 molecules with the PN binding site exposed outside. Mostly, these kinetics were similar to that measured with external ADP or ATP, which reflects the opposite UCP orientation.

The Eadie-Hofstee plot for the experiment with externally added ADP is nearly parallel to the control plot (Fig. 2a). Thus, kinetics in the absence and presence of ADP exhibits almost equal Km, while Vmax with ADP was about half of the control. As with UCP1, this fact reflects a noncompetitive and perhaps an allosteric type of nucleotide inhibition. Although subject to errors, the differential rates also yielded an Eadie-Hofstee plot parallel with those for control or ADP. The apparent Km values for lauric acid and UCP2 or UCP3 (Table I) were higher than those found for UCP1 (22). Note, that this has been also observed for CoQ10-activated E. coli-expressed UCP2 and UCP3 (21).

Kinetics of UCP2-mediated H+ Efflux Induced by Naturally Abundant Fatty Acids—Among naturally abundant FAs, myristic acid, palmitic acid (Fig. 2b), oleic acid (Fig. 2c), linoleic acid, and arachidonic acid (Fig. 2d) exhibited the ability to induce H+ efflux in proteoliposomes containing UCP2 (Table I) or UCP3 (not shown). The saturated increase of the total H+ fluxes with the increasing total FAs was observed for all FAs tested. When comparing H+ flux densities per square micrometer (39) (Table I), the densities in UCP2 or UCP3 proteoliposomes were much higher than the densities for background H+ fluxes in protein-free liposomes. They were usually also higher than the H+ flux densities estimated in the vesicles containing extracts from W303 yeast mitochondria (Table I). The Eadie-Hofstee plots constructed for total H+ fluxes in micromoles of H+·min-1·(mg of protein)-1 were again parallel with those for H+ fluxes in the presence of external ATP or ADP and largely similar differential rates (dotted lines in Fig. 2, a–d).

The apparent affinity for various FAs (taken as the inverse Km, Table I) was quite similar but decreased in order for heptylbenzoic > palmitic > lauric > linoleic > arachidonic > oleic > myristic acid. Note that PUFAs (vide infra) and non-physiological heptylbenzoic acid exhibited higher affinity than naturally abundant FAs. The Vmax values and turnover numbers per dimer are also listed in Table I. The turnovers represent the minimum estimates, since not all measured protein is likely to be active. Among abundant FAs, the highest turnover was found for oleic acid, while it decreased for myristic > linoleic > arachidonic >= palmitic >= lauric acid. Heptylbenzoic acid exhibited the lowest evaluated turnover. Only 10% H+ flux was observed for caprylic acid and less than 5% for 12-hydroxylauric acid, an inactive FA which is unable to flip-flop across the lipid bilayer (46). Note that H+ fluxes in the absence of FAs amounted to 7–10% of Vmax for lauric acid (Table I). Similar results were found with UCP3 (the apparent Km values were 170, 260, and 197 µM for lauric, myristic, and oleic acid, respectively (Vmax values were 147, 286, and 343.10-6 pmol of H+ µm-2).

Do Exceptional Activating Fatty Acids Exist for UCP2?— Among the naturally abundant FAs tested as the above initial series, we have found almost no "exceptional" FAs that would induce significantly higher H+ fluxes in UCP2 proteoliposomes or a high apparent affinity, given by a very low Km. Table I indicates that oleic and myristic acid exhibited the highest rates, but also a quite high Km, reflecting the low affinity. Unsaturation was not a major factor, since arachidonic acid (C20:4 {omega}-6) behaved similarly to palmitic and lauric acid. Nevertheless, we found more potent FAs when the kinetics of PUFAs was tested. Thus, cis-8,11,14-eicosatrienoic (or dihomo-{gamma}-linolenic, C20:3 {omega}-6) and cis-6,9,12 octadecatrienoic ({gamma}-linolenic, C18:3 {omega}-6) were found to induce very fast H+ efflux in UCP2 proteoliposomes. However, their kinetics exhibited the lowest Km values found: 20 and 29 µM, respectively (Fig. 3 and Table I). Similar results were found for UCP3. Also, cis-5,8,11,14,17-eicosapentaenoic (EPA, C20:5 {omega}-3, Table I) and cis-4,7,10,13,16,19 docosahexaenoic acid (C22:6 {omega}-3) were more efficient than FAs of the basic series of naturally abundant FAs. EPA exhibited the third highest apparent affinity (Table I).

No Effect of Coenzyme Q10 on UCP2- and UCP3-mediated H+ Efflux Induced by Lauric Acid—We have also tested the effect of the presumed activating cofactor, coenzyme Q10. No effect of oxidized CoQ10 (1–5 µM) was observed when added directly to the assay (Fig. 1a, right panel), nor when oxidized CoQ10 was added to lipids during extraction of yeast mitochondria and formation of vesicles (not shown). This indicates that in our reconstituted system the recombinant yeast-expressed UCP2 and UCP3 are intact and do not need further activation other than by FAs. These results, however, do not entirely exclude the existence of CoQ10 activation of UCPs, since the effective CoQ10 dose could be extracted from yeast mitochondria. Nevertheless, with E. coli-expressed proteins CoQ10 activation did not exceed 30%: 1 µM CoQ10 at 100 µM lauric acid activated UCP2 to 132%; 0.1 µM CoQ10 activated UCP2 to 103%, UCP3 to 135%.4

Nucleotide Inhibition of UCP2 and UCP3—Also the above kinetic data demonstrated that the observed H+ fluxes were up to 50% inhibited by various PN di- and triphosphates.4 The inability of complete inhibition by the external PN can be explained by two equally distributed orientations of UCP molecules in the membrane, with PN binding sites exposed outside and inside (22). Hence, we could evaluate the inhibitory dose responses for lauric acid-induced H+ uniport while assuming a decrease to a 50% rate as 100% inhibition. Stock nucleotide solutions that are usually acidic had to be buffered by Tris base, and assay pH was carefully maintained at 7.2. Otherwise, artificial "inhibition" was observed because of a decrease in rate by acidic pH. For reconstituted UCP2, the lowest Ki was exhibited by ADP (350 µM, Fig. 4), while ATP exhibited higher Ki (445 µM) reflecting a slightly lower affinity of ATP to UCP2 (Fig. 4). These results were confirmed by three independent experiments. The apparent affinity for GTP was slightly lower than for ATP (Table II) and for GDP it was even lower (Table II). The lowest affinity was found for AMP (Fig. 4 and Table II); indeed it is similar to the low affinity of AMP and GMP versus ATP or GTP, previously documented for UCP1 (51, 52). Similar results were obtained with UCP3 (Table II). The magnitudes of Ki values for PN inhibition are higher than those found for UCP1, as also tested with recombinant yeast-expressed UCP1 (Table II), but are lower than those reported for E. coli-expressed protein (20).



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FIG. 4.
Nucleotide dose responses for inhibition of lauric acid-induced H+ fluxes in UCP2 proteoliposomes by ADP ({square}), ATP ({blacktriangledown}), and AMP (•). Inhibition of H+ fluxes induced by 200 µM lauric acid in proteoliposomes containing UCP2 was measured and dose responses were constructed on the assumption of maximum inhibition at a 50% rate (main panel) from the data expressed in relative rates (inset). Solid lines represent fits by the Hill equation, which yielded the inhibitory constant Ki values 350 µM for ADP, 445 µM for ATP, and 3140 µM for AMP.

 

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TABLE II
Inhibitory constants for nucleotide inhibition of UCPs

 

Binding of [3H]GTP and [3H]ATP to Isolated UCP2 and UCP3—Fig. 5 illustrates the typical Scatchard plots for [3H]GTP and [3H]ATP binding to yeast-expressed recombinant UCP2 preparation, while Table III summarizes the results of several experiments. [3H]GTP binding in the presence of 1 µM CAT is also shown in Fig. 5, demonstrating binding under conditions eliminating possible contribution of the ADP/ATP carrier (CAT prevents nucleotide binding to this carrier and GTP has a much lower affinity to it). The observed saturated binding was partially prevented by the unlabeled (cold) GTP or ATP, respectively, but [3H]GTP binding was also prevented by ATP and ADP (not shown). Binding constant Kd values amounting to the average of ~1.5 µM (Table III) were derived from the Scatchard plots for both, the GTP-sensitive portion of [3H]GTP binding, and the ATP-sensitive part of [3H]ATP binding. Taking into account the estimated protein content and assuming UCP2 as 100% pure, the calculated number of binding sites corresponded to 1.29 or 0.85 per UCP2 monomer without or with CAT, respectively (Fig. 5). Kd for the experiment of Fig. 5 was 1.8 µM without CAT (a higher value could reflect contribution of the ADP/ATP carrier) and 1.68 µM with CAT. For [3H]ATP, Kd changed from 1.5 µM (without CAT, Fig. 5, Table III) to 1.3 µM with CAT, whereas the apparent number of binding sites decreased from 1.5 per UCP2 monomer by ~10–20%.



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FIG. 5.
3H-labeled nucleotide binding to isolated yeast-expressed UCP2. ({square}), [3H]GTP in the absence of CAT; ({blacksquare}),[3H]GTP in the presence of 1 µM CAT; ({circ}), [3H]ATP in the absence of CAT. Yeast-expressed UCP2 was isolated and assayed with radiolabeled PN as described under "Experimental Procedures." Scatchard plots are shown for two parallel measurements with subtracted data for background measured with 2.5 mM cold GTP or ATP, respectively. The derived binding constant Kd values were 1.8 µM for [3H]GTP without CAT; 1.68 µM for [3H]GTP with CAT; and 1.46 µM for [3H]ATP (single experiment). Assuming that preparation contains only a UCP2 protein (Mr ~33,000), the calculated numbers of [3H]GTP binding sites corresponded to 1.29 per UCP2 monomer (1.5 for [3H]ATP) in the absence of CAT and to 0.85 per UCP2 monomer in the presence of CAT.

 

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TABLE III
Nucleotide binding constants for recombinant UCP2 and UCP3

 

Similar data but with lower affinities (higher Kd values) and lower numbers of binding sites were obtained for UCP2 and UCP3 expressed in E. coli (Fig. 6 and Table III). CAT had no effect (Fig. 6), thus reflecting that no ADP/ATP carrier contamination is possible with this expression (as well as no Coenzyme Q carryover). Despite this, the obtained Kd values were three times higher, possibly also caused by the remaining lauroylsarcosinate present. We can conclude that the observed saturated binding reflects the nucleotide binding sites of recombinant UCP2 and UCP3 in both cases of expression. The higher affinity and higher proportion of binding sites obtained for yeast-expressed proteins reflect the advantage of this system and the lipid protection used.



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FIG. 6.
[3H]GTP nucleotide binding to isolated E. coli-expressed UCP2 or UCP3. ({diamond}), UCP2 in the absence of CAT; ({circ}), UCP2 with 1 µM CAT; ({blacktriangledown}), UCP3. E. coli-expressed UCP2 and UCP3 were isolated and assayed with radiolabeled PN as described under "Experimental Procedures." Scatchard plots are shown with subtracted data for background measured with 2.5 mM cold GTP. The derived binding constant Kd values are 5.8 µM for UCP2 in the absence of CAT, 5.1 µM in the presence of 1 µM CAT (fit by the dotted line), and 7.27 µM for UCP3, while the calculated number of binding sites corresponded to 0.4 and 0.35 per UCP2 monomer in the absence or presence of 1 µM CAT, respectively, and to 0.35 per UCP3 monomer.

 

[3H]GTP Binding to Isolated Mitochondria of Several Tissues—In order to demonstrate the existence of native UCPs in intact tissues and to show the relevancy of the binding method not only for recombinant, but also for native proteins, we have evaluated [3H]GTP binding in the presence of CAT in mitochondria isolated from rat liver, skeletal muscle, kidney, and lung (Fig. 7). In agreement with the findings of Pecqueur et al. (19), we found the highest number of [3H]GTP binding sites in lung mitochondria (182 ± 18 pmol/mg of protein). This is about four times lower than the usual amount of [3H]GTP binding sites reflecting mostly the UCP1 molecules in BAT mitochondria (53). However, the evaluated Kd of 0.43 ± 0.03 µM reflects an even higher affinity than found with yeast-expressed human UCP2. The estimated total number of [3H]GTP binding sites was lower (74 ± 22 and 28 ± 6 pmol/mg of protein, Kd values of 0.3 ± 0.06 µM and 0.14 ± 0.02 µM) in kidney and skeletal muscle mitochondria, respectively, accounting for~10 and ~30 times less than for UCP1 in BAT mitochondria. The lowest amount was found in liver mitochondria (21 ± 4 pmol/mg of protein; Kd 0.23 ± 0.03 µM). The measured proportions between the numbers of [3H]GTP binding sites in the studied tissues correlate well to the proportions of UCP2 mRNA typically found in these tissues (5, 6, 7, 19). Hence, even if not all [3H]GTP binding sites could be ascribed to the native UCP2 (UCP2 and UCP3 in skeletal muscle), the interfering part should represent a minor portion. Consequently, these data represent the first demonstration of the existence of high affinity nucleotide binding to native UCP2 (UCP3).



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FIG. 7.
[3H]GTP binding to isolated mitochondria from several rat tissues. ({circ}), lung; ({blacktriangleup}), kidney; ([itrio), skeletal muscle; ({blacktriangledown}), liver. Scatchard plots are shown with subtracted data for background, measured with 2.5 mM cold GTP. Average values ± SDs are plotted for data measured on several mitochondrial isolations (n). The derived number of binding sites corresponded to 182 ± 18 pmol/mg of protein, n = 4, in lung; to 74 ± 22 (n = 3), 28 ± 6 (n = 2), and 21 ± 4 pmol/mg of protein (n = 5), in kidney, skeletal muscle, and liver mitochondria, respectively. ± errors of x-intercepts were taken from linear regression fits. The derived binding constant Kd values were 0.43 ± 0.03 µM for lung; 0.3 ± 0.06 µM, 0.14 ± 0.02 µM, and 0.23 ± 0.03 µM in kidney, skeletal muscle, and liver mitochondria, respectively.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this work we have evaluated in detail the possible phenotypes of the novel uncoupling proteins UCP2 and UCP3. We have identified their best up-to-date known activating and inhibitory ligands. We have demonstrated that two (C18 and C20) {omega}-6 PUFAs are the most potent activators of UCP2 (UCP3), whereas among purine nucleotides, ADP is the most potent inhibitor. We have for the first time demonstrated the binding of natural 3H-labeled nucleotides to recombinant UCP2 (UCP3) proteins and to the mitochondria of several tissues.

Concerning activating FAs, we have clearly demonstrated that all physiologically abundant long chain FAs, saturated or unsaturated, activate H+ translocation in UCP2 and UCP3 proteoliposomes. This is demonstrated by the parameters of their activating (FA cycling) kinetics. We cannot explain why Rial et al. (36) could not find any response with their tested FAs, except for the all-trans-retinoic acid in yeast mitochondria of yeast-expressing UCP2. Among our tested FAs, we have found that oleic acid exhibited the highest rate, but {omega}-6 PUFAs, such as cis-8,11,14-eicosatrienoic (C20:3 {omega}-6) and cis-6,9,12 octadecatrienoic (C18:3 {omega}-6) exhibited both high Vmax and the highest apparent affinity (1/Km). As such they were very efficient in inducing H+ uniport mediated by UCP2. Hence, we have shown that more efficient FAs do indeed exist and are activating UCP2-mediated H+ uniport in lower concentrations (amounts) than that required for other natural FAs. Their low in vivo abundance is balanced by their high activating profile. This is valid for the two {omega}-6 PUFAs, which were identified by us as the best UCP2 activators. For example, the typical content as 1.9 and 2.9 µg of C20:3 {omega}-6, or 0.8 and 0.4 µg of C18:3 {omega}-6, was identified per mg of phospholipids in rat liver and kidney, respectively (54). The amount of C20:3 {omega}-6 in human plasma phospholipids is equivalent to 100 µM (55). One can speculate that if 10% of these amounts would be cleaved off, a substantial activation of UCP2 will occur. A slightly lower efficiency with regard to UCP2 was found for {omega}-3 PUFAs, EPA (C20:5 {omega}-3) and docosahexaenoic acid (C22:6 {omega}-3), but because of the very high content of the latter in the brain or retina tissues (56), activation of UCP2 by C22:6 {omega}-3 is very plausible. The efficiency of C20:5 {omega}-3 and C22:6 {omega}-3 was still higher than that of arachidonic or oleic, or lauric and palmitic acid (equally effective as lauric). Our results have a great physiological relevance, since {omega}-6 PUFAs are among the most efficient activators of PPAR{beta} (17) and together with {omega}-3 PUFAs also to potent activators of PPAR{gamma} (18) and PPAR{alpha} (57). In conclusion, our findings suggest their possible dual role in activating both UCP2 (UCP3) expression and the uncoupling activity.

Our results have also shown that protonophoric phenotypes of UCP2 and UCP3 do not differ qualitatively from the UCP1 phenotype. The per dimer turnovers (estimated from Vmax and total protein) fall into the range of hundreds per second (Table II). It is similar to the turnover of 94 s-1 that can be derived from the rate of 86.7 µmol of H+ min-1(mg of protein)-1 for lauric acid at 25 °C and CoQ10-activated E. coli expressed UCP2 (21). Also, the maximum per dimer turnover reported for UCP1 (133 s-1, Ref. 26) is comparable. The same is true for plant UCP (99 s-1, Ref. 47). The situation is more complex when one compares experimental apparent affinities taken as 1/Km. Since, we did not subtract a contribution of non-protein-related H+ leak, the real Km values could be lower. Our Km values (except for a few or those for PUFAs) are higher than those reported for UCP1 and for the phosphate carrier (49). Nevertheless, similar high Km values can be derived from the results reported by Klingenberg and co-workers (21).

Concerning PN inhibition, the derived Ki values for UCP2 and UCP3 were higher than those for UCP1 in our reconstituted system, but all Ki values for ATP and ADP were within the range of several hundred micromolar. We must admit that sulfate used in our assay media strongly decreases the PN affinity to UCP1 (25) and presumably also to UCP2/UCP3, which might explain the discrepancies between our Ki values and those measured by Klingenberg and co-workers (21, 58) and partly also between the Ki values (Table II) and binding constant Kd values (Table III). We chose sulfate to ensure that the anion used would be membrane impermeant, would not quench the SPQ probe, and that UCPs would not transport it. Thus, sacrificing the efficiency of nucleotide inhibition, we ensured high K+ diffusion potential and the correct probe response. Our medium may better simulate in vivo conditions. We have also demonstrated that ADP is a slightly stronger UCP2 inhibitor than ATP. The same finding has previously been reported for UCP3 (58). GTP and GDP exhibited a slightly lower inhibitory ability. However, the differences between ATP and GTP were not found for the binding constant Kd. It is also not certain whether the observed small difference in ATP and ADP affinities in vitro can account for the suggested in vivo activation of UCP3 by the increased ATP/ADP ratio (58) simply because of higher ADP affinity.

We have also confirmed the existence of PN interaction with UCP2 (UCP3) by direct binding experiments using [3H]GTP and [3H]ATP. Despite the fact that the binding of fluorescent nucleotide derivatives was reported recently (28), our results represent the first measurements with natural PNs (Figs. 5, 6, 7). We have determined equal binding constant Kd values and nearly an equal number of binding sites for [3H]GTP and [3H]ATP for yeast-expressed proteins. Also, the number of PN binding sites was not reduced to zero with CAT and actually amounted roughly to 1 with CAT. This indicates that if binding to the ADP/ATP carrier contributes to the total binding in the samples obtained by yeast expression, it represents a minor contribution. The data obtained with CAT then reflect net binding to UCP2. For E. coli-expressed UCP2 and UCP3, similar data were found, but with higher Kd values and lower binding site numbers. This may reflect the continued presence of interfering lauroylsarcosinate or that only a portion of the population of UCPn molecules has an intact conformation. Nevertheless, the affinity derived as reciprocal Kd is about three times higher for the native UCP2 in mitochondria than for recombinant yeast-expressed UCP2. Obviously, the natural insertion of UCP2 molecule in the internal membrane is superior to the micellar solution.

The existence of high affinity [3H]GTP binding sites in mitochondria suggests that these sites may indeed reflect the binding to native UCP2 (UCP2 plus UCP3 in skeletal muscle mitochondria). This conclusion is strongly supported by our finding of the highest number of [3H]GTP binding sites in the mitochondria of tissues where UCP2 content was reported to be high, i.e. in lung (19) and kidney (27). On the contrary, we found a ~10 times lower number of [3H]GTP binding sites in the mitochondria of liver, an organ which has almost no UCP2 expression (6, 7), unless it is stimulated, e.g. by cytokines (2, 9). This fact again suggests that the detected [3H]GTP binding sites could be predominantly formed by UCP2. Surprisingly, skeletal muscle mitochondria, presumably containing both UCP2 and UCP3, exhibited only a slightly higher content of [3H]GTP binding sites than liver mitochondria. However, this estimate fits well with protein measurements using antibodies (19). In conclusion, the maximum UCP2 content found in lung mitochondria (~200 pmol/mg of protein, Fig. 7) is about four times less abundant than the content of UCP1 in BAT mitochondria (53). In skeletal muscle, the UCP2 + UCP3 content derived from the total number of [3H]GTP binding sites was ~7 times smaller than the UCP2 in the lung, and in the liver, UCP2 is even less abundant. The finding of small amounts of UCP2 in isolated mitochondria supports the concept of UCP2 (UCP3) as a stress protein, which can be elevated as required under various physiological situations (2, 5).

It would seem to be trivial to note that no superoxide activation was required in our experiments to demonstrate PN binding or FA-activated H+ uniport. But, since other investigators claim that only under such activation can PN inhibition of FA-induced uncoupling be observed (27), we must stress this point. In conclusion, we have demonstrated that yeast-expressed UCP2 and UCP3 are able to mediate PN-sensitive H+ transport in the presence of FAs and that recombinant yeast- or E. coli-expressed UCP2 and UCP3 do indeed bind purine nucleotides. The latter property is reflected in vivo by the various number of PN binding sites in the mitochondria of various tissues. Their number, and hence the amount of UCP2 (UCP3) is, however, quite low. Except for lung mitochondria, it is at least 10 times lower than the amount of UCP1 in BAT mitochondria. Despite this fact, in vivo, when not all FAs are metabolized or when FAs would be cleaved off lipids (e.g. by phospholipase C), a certain non-zero uncoupled state may exist in all types of mitochondria. Such a weak uncoupling state would be enabled by the lowering (modulating) PN affinity due to elevated concentrations of anions, Mg2+, or other putative modulators of PN inhibition. The resulting weak uncoupling also prevents the excessive production of reactive oxygen species. Yet, the unknown regulatory mechanisms may further release PN inhibition to achieve a higher or complete activation of UCPn-mediated uncoupling. The states of higher uncoupling would then represent the specific thermogenic roles of UCPn in different tissues, such as tissue-specific adaptive thermogenesis, and simultaneously, a state with a very low mitochondrial production of the reactive oxygen species.


    FOOTNOTES
 
* This work was supported by Research Project AVOZ5011922, by grants from the program Kontakt from the Czech Ministry of Education (ME 389), Grant Agency of the Czech Republic, (No. 301/02/1215), and by the Internal Grant Agency of the Academy of Sciences of the Czech Republic (No. A50111 [GenBank] 06). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. No.75, Membrane Transport Biophysics, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 14220 Prague 4, Czech Republic. Tel.: 011-420-296442760; Fax: 011-420-296442488; E-mail: jezek{at}biomed.cas.cz.

1 The abbreviations used are: UCP2, ubiquitous uncoupling protein; BAT, brown adipose tissue; CAT, carboxyatractyloside; EPA, cis-5,8,11,14,17-eicosapentaenoic (C20:5 {omega}-3) acid; FA, fatty acid; HTP, hydroxylapatite; MES, 2-[N-morpholino] ethanesulfonic acid; Octyl-POE, octylpentaoxyethylene; PN, purine nucleotides; UCP1, uncoupling protein of brown adipose tissue mitochondria; UCP3, skeletal muscle-specific uncoupling protein; UCPn, any uncoupling protein; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; TEA, tetraethyl ammonium; TES, N-Tris [hydroxymethyl]methyl-2-amino-ethanesulfonic acid; CoQ10, coenzyme Q10; PPAR, peroxisome proliferator-activated receptor; PUFA, polyunsaturated FA; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight. Back

2 At much lower lipid-to-protein ratio in much larger vesicles, about 6 proteins per vesicle were estimated by Winkler, E., and Klingenberg, M. (1992) Eur. J. Biochem. 207, 135–145. Back

3 Unpublished data of P. Jezek and M. Zacková have shown inhibition of fatty acid cycling via ADP/ATP carrier by agaric acid. Back

4 P. Jezek, E. Skobisová, and M. Zacková, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank the excellent technical assistance of Jana Brucknerová and Jana Kosarová as well as the help of Petr Hanák, M. S. with yeast expression and of Tomás Spacek, M. S. with some binding assays. The fluorimeter was purchased from the funds of the Czech-U.S. Science and Technology Program, Grant No. 86043.



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 DISCUSSION
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