Native UCP1 Displays Simple Competitive Kinetics between the Regulators Purine Nucleotides and Fatty Acids

Elucidation of the regulation of uncoupling protein 1 (UCP1) activity in its native environment, i. e. the inner membrane of brown-fat mitochondria, has been hampered by the presence of UCP1-independent, quantitatively unresolved effects of investigated regulators on the brown-fat mitochondria themselves. Here we have utilized the availability of UCP1-ablated mice to dissect UCP1-dependent and UCP1-independent effects of regulators. Using a complex-I-linked substrate (pyruvate), we found that UCP1 can mediate a 4-fold increase in thermogenesis when stimulated with the classical positive regulator fatty acids (oleate). After demonstrating that the fatty acids act in their free form, we found that UCP1 increased fatty acid sensitivity ∼30-fold (as compared with the 1. 5-fold increase reported earlier based on nominal fatty acid values). By identifying the UCP1-mediated fraction of the response, we could conclude that the interaction between purine nucleotides (GDP) and fatty acids (oleate) unexpectedly displayed simple competitive kinetics. In GDP-inhibited mitochondria, oleate apparently acted as an activator. However, only a model in which UCP1 is inherently active (i. e. “activating” fatty acids cannot be included in the model), where GDP functions as an inhibitor with a Km of 0. 05 mm, and where oleate functions as a competitive antagonist for the GDP effect (with a Ki of 5 nm) can fit all of the experimental data. We conclude that, when examined in its native environment, UCP1 functions as a proton (equivalent) carrier in the absence of exogenous or endogenous fatty acids. Elucidation of the regulation of uncoupling protein 1 (UCP1) activity in its native environment, i. e. the inner membrane of brown-fat mitochondria, has been hampered by the presence of UCP1-independent, quantitatively unresolved effects of investigated regulators on the brown-fat mitochondria themselves. Here we have utilized the availability of UCP1-ablated mice to dissect UCP1-dependent and UCP1-independent effects of regulators. Using a complex-I-linked substrate (pyruvate), we found that UCP1 can mediate a 4-fold increase in thermogenesis when stimulated with the classical positive regulator fatty acids (oleate). After demonstrating that the fatty acids act in their free form, we found that UCP1 increased fatty acid sensitivity ∼30-fold (as compared with the 1. 5-fold increase reported earlier based on nominal fatty acid values). By identifying the UCP1-mediated fraction of the response, we could conclude that the interaction between purine nucleotides (GDP) and fatty acids (oleate) unexpectedly displayed simple competitive kinetics. In GDP-inhibited mitochondria, oleate apparently acted as an activator. However, only a model in which UCP1 is inherently active (i. e. “activating” fatty acids cannot be included in the model), where GDP functions as an inhibitor with a Km of 0. 05 mm, and where oleate functions as a competitive antagonist for the GDP effect (with a Ki of 5 nm) can fit all of the experimental data. We conclude that, when examined in its native environment, UCP1 functions as a proton (equivalent) carrier in the absence of exogenous or endogenous fatty acids. Uncoupling protein 1 (UCP1) 1The abbreviations used are: UCP1, uncoupling protein 1; TES, 2-2-hydroxy-1, 1-bis (hydroxymethyl) ethylaminoethanesulfonic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. is essential for the phenomenon of classical nonshivering thermogenesis (1Nedergaard J. Golozoubova V. Matthias A. Asadi A. Jacobsson A. Cannon B. Biochim. Biophys. Acta. 2001; 1504: 82-106Crossref PubMed Scopus (468) Google Scholar, 2Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4568) Google Scholar). When chronically exposed to cold, animals that can recruit UCP1 in their brown-adipose tissue will initially shiver but will then successively increase their capacity for nonshivering thermogenesis and ultimately cease shivering. Mice that lack UCP1 (3Enerbäck S. Jacobsson A. Simpson E. M. Guerra C. Yamashita H. Harper M. -E. Kozak L. P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1089) Google Scholar) never cease shivering in the cold (4Golozoubova V. Hohtola E. Matthias A. Jacobsson A. Cannon B. Nedergaard J. FASEB J. 2001; 15: 2048-2050Crossref PubMed Scopus (360) Google Scholar). Thus, no other process or protein can replace UCP1 in its physiological thermogenic function. Brown-fat cells from animals that can express UCP1 respond to stimulation with the physiological activator norepinephrine with a increase in the of Cannon B. J. PubMed Scopus Google Scholar, A. Jacobsson A. Nedergaard J. Cannon B. J. PubMed Scopus Google Scholar) and in the of thermogenesis J. Cannon B. Nature. PubMed Scopus Google Scholar). In is no effect of norepinephrine on in brown-fat cells that lack UCP1 A. Jacobsson A. Nedergaard J. Cannon B. J. PubMed Scopus Google Scholar). Thus, UCP1 is essential for the of brown-fat cells to fatty acids can to the as can norepinephrine in brown-fat cells Cannon B. J. 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J. 2001; PubMed Scopus Google of the with the and with on the and included in the mice free to and on a in between the and mice used for the by the of the mitochondria as B. PubMed Scopus Google Scholar) with on experimental 5 UCP1-ablated mice for by a of and and and brown-adipose tissue and from and tissue and in the the tissue tissue with in of and for with the in fatty and for then for and the in protein the and the to of of of with fatty on and used for no mitochondria, a of of to of a of 1 fatty and of with a in a from the and by a to the and to based on an of of of on the and on the of protein of the of from as the in in an and the experimental for substrate 5 free of oleate the used by A. PubMed Scopus Google Scholar) for the of oleate to fatty where is the of oleate to the in the nominal of the oleate and Thus, to free of and only the free purine or the is for to UCP1 J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, Biochim. Biophys. Acta. 2001; 1504: PubMed Scopus Google the for free GDP used in and for of GDP and in the and by of a for of a PubMed Scopus Google Scholar). nominal of GDP and to the free of and nominal of to a free of in the presence of all of the and 1 of free to in all of the experimental with GDP by a of to the with the fit of the for for to simple the effect of with a of used for in with of fatty on and A. PubMed Scopus Google Scholar). fraction from acid cyanide GDP acid acid and all from GDP in and the in from from in in in and in a of in have effects on the of the as the for the of of UCP1 on earlier of the UCP1-dependent of thermogenesis in brown-fat mitochondria, used as substrate A. Jacobsson A. Cannon B. Nedergaard J. J. PubMed Scopus Google Scholar, S. Kozak L. P. Harper J. Physiol. Google Scholar, Harper Kozak L. P. J. 2001; PubMed Scopus Google Scholar, J. Golozoubova V. Matthias A. Ki Jacobsson A. Cannon B. 2001; PubMed Google Scholar). the effects of the presence of UCP1 quantitatively when the effects of fatty acids UCP1-dependent effects when the used with as but the with In is that in brown-fat mitochondria UCP1 the and the of GDP effect but could be increased by In brown-fat mitochondria with UCP1 the of and by However, the sensitivity of the mitochondria to by the presence or absence of UCP1 and of UCP1 for in oleate could in brown-fat mitochondria UCP1 but the effect of oleate oleate and in brown-fat mitochondria with UCP1 the in is that the presence of UCP1 to a sensitivity to from an of 5 to an of i. e. a However, that, in the mitochondria with UCP1, fatty acids (oleate) could an increase in the of that i. e. a increase to the fatty increase in in brown-fat cells A. Jacobsson A. Nedergaard J. Cannon B. J. PubMed Scopus Google Scholar). In the fatty increase in when used as substrate only in with earlier A. Jacobsson A. Cannon B. Nedergaard J. J. 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Acta. 2001; 1504: 82-106Crossref PubMed Scopus (468) Google but of have UCP1-independent the of the UCP1-dependent In brown-fat mitochondria UCP1, the for oleate stimulation of when the of GDP increased a effect when stimulated with of However, in brown-fat mitochondria with UCP1, the for oleate by the GDP Km increased from to in the presence of 1 the in stimulation of interaction between oleate and GDP in that the oleate sensitivity all in with the of a purine interaction with UCP1 but no interaction with nucleotides Cannon B. of Scholar). interaction for oleate and UCP1, the for in mitochondria with UCP1 only by GDP and to the as the for mitochondria UCP1 of the between and that the interaction between purine nucleotides and fatty acids could be when the UCP1-dependent fraction of the we examined the interaction between fatty acids and purine nucleotides in in to increase the of the regulation of UCP1 activity its of have been all effects of purine nucleotides and fatty acids on UCP1 as by Nedergaard (1Nedergaard J. 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