title: Uncoupling protein-1 is not leaky.
abstract: The activity of uncoupling protein-1 (UCP1) is rate-limiting for nonshivering thermogenesis and diet-induced thermogenesis. Characteristically, this activity is inhibited by GDP experimentally and presumably mainly by cytosolic ATP within brown-fat cells. The issue as to whether UCP1 has a residual proton conductance even when fully saturated with GDP/ATP (as has recently been suggested) has not only scientific but also applied interest, since a residual proton conductance would make overexpressed UCP1 weight-reducing even without physiological/pharmacological activation. To examine this question, we have here established optimal conditions for studying the bioenergetics of wild-type and UCP1-/- brown-fat mitochondria, analysing UCP1-mediated differences in parallel preparations of brown-fat mitochondria from both genotypes. Comparing different substrates, we find that pyruvate (or palmitoyl-L-carnitine) shows the largest relative coupling by GDP. Comparing albumin concentrations, we find the range 0.1-0.6% optimal; higher concentrations are inhibitory. Comparing basic medium composition, we find 125 mM sucrose optimal; an ionic medium (50-100 mM KCl) functions for wild-type but is detrimental for UCP1-/- mitochondria. Using optimal conditions, we find no evidence for a residual proton conductance (not a higher post-GDP respiration, a lower membrane potential or an altered proton leak at highest common potential) with either pyruvate or glycerol-3-phosphate as substrates, nor by a 3-4-fold alteration of the amount of UCP1. We could demonstrate that certain experimental conditions, due to respiratoty inhibition, could lead to the suggestion that UCP1 possesses a residual proton conductance but find that under optimal conditions our experiments concur with implications from physiological observations that in the presence of inhibitory nucleotides, UCP1 is not leaky.
authors.affiliation: The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Irina G
authors.initials: IG
authors.lastname: Shabalina
authors.forename: Mario
authors.initials: M
authors.lastname: Ost
authors.forename: Natasa
authors.initials: N
authors.lastname: Petrovic
authors.forename: Marek
authors.initials: M
authors.lastname: Vrbacky
authors.forename: Jan
authors.initials: J
authors.lastname: Nedergaard
authors.forename: Barbara
authors.initials: B
authors.lastname: Cannon
mesh_terms: D002001:Adipose Tissue, Brown / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000187:drug effects / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002417:Cattle; D003470:Culture Media; D004563:Electrochemistry; D004734:Energy Metabolism / Q000187:drug effects; D005994:Glycerophosphates / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D011522:Protons; D019289:Pyruvic Acid / Q000378:metabolism; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins / Q000187:drug effects / Q000378:metabolism; D012710:Serum Albumin, Bovine; D019802:Succinic Acid / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology; D000071256:Uncoupling Protein 1
chemical_list: D051017:Apoptosis Regulatory Proteins; D003470:Culture Media; C097041:DAP3 protein, human; D005994:Glycerophosphates; D007473:Ion Channels; D024101:Mitochondrial Proteins; D011522:Protons; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C000604371:UCP1 protein, human; C000604372:Ucp1 protein, mouse; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D006153:Guanosine Diphosphate; D012710:Serum Albumin, Bovine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D019289:Pyruvic Acid; C029620:alpha-glycerophosphoric acid; D019802:Succinic Acid