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 similar to30-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 K-m of 0.05 mM, and where oleate functions as a competitive antagonist for the GDP effect (with a K-i 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.