Activating ω-6 polyunsaturated fatty acids and inhibitory purine nucleotides are high affinity Ligands for novel mitochondrial uncoupling proteins UCP2 and UCP3

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 Q(10) had no further significant activating effect. Evaluated parameters of FA activation (FA cycling) kinetics revealed the highest apparent affinity to UCP2 ( the lowest K-m values: 20 and 29 muM, 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 PPARbeta receptor in the activation of UCP2 transcription. omega-3 PUFA, cis-5,8,11,14,17-eicosapentaenoic acid had lower affinity (K-m, 50 muM), although as an omega-6 PUFA, arachidonic acid exhibited the same low affinity as lauric acid (K-m, similar to200 muM). 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 K-i) decreasing in order: ADP>ATPsimilar toGTP>GDPmuch greater thanAMP. Also [H-3] GTP ([H-3] ATP) binding to isolated Escherichia coli (K-d, similar to5 muM) or yeast-expressed UCP2 (K-d, similar to1.5 muM) or UCP3 exhibited high affinity, similar to UCP1. The estimated number of [H-3] GTP high affinity (K-d, <0.4 mu M) binding sites was (in pmol/mg of protein) 182 in lung mitochondria, 74 in kidney, 28 in skeletal muscle, and similar to 20 in liver mitochondria. We conclude that purine nucleotides must be the physiological inhibitors of UCPn-mediated uncoupling in vivo.