In isolated brown adipose tissue mitochondria, UCP1 is not essential for - nor involved in - the uncoupling effects of the classical uncouplers FCCP and DNP

• Published in: Biochimica et biophysica acta. Bioenergetics 2025-01, Vol.1866 (1), p.149516, Article 149516
• Main Authors: Shabalina, Irina G., Jiménez, Beatriz, Sousa-Filho, Celso Pereira Batista, Cannon, Barbara, Nedergaard, Jan
• Format: Article
• Language: English
• Subjects: 2,4-dinitrophenol (DNP); Adipose Tissue, Brown - metabolism; Animals; brown adipose tissue mitochondria; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone - pharmacology; Energy Metabolism - drug effects; FCCP; Ion Channels - metabolism; Membrane potential; Methyl-β-cyclodextrin; Mice; Mice, Knockout; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Oxygen Consumption - drug effects; Uncoupling Agents - pharmacology; Uncoupling protein 1 (UCP1); Uncoupling Protein 1 - genetics; Uncoupling Protein 1 - metabolism
• ISSN: 0005-2728; 1879-2650; 1879-2650
• DOI: 10.1016/j.bbabio.2024.149516

Recent patch-clamp studies of mitoplasts have challenged the traditional view that classical chemical uncoupling (by e.g. FCCP or DNP) is due to the protonophoric property of these substances themselves. These studies instead suggest that in brown-fat mitochondria, FCCP- and DNP-induced uncoupling is mediated through activation of UCP1 (and in other tissues by activation of the adenine nucleotide transporter). These studies thus advocate an entirely new paradigm for the interpretation of standard bioenergetic experiments. To examine whether these patch-clamp results obtained in brown-fat mitoplasts are directly transferable to classical isolated brown-fat mitochondria studies, we investigated the effects of FCCP and DNP in brown-fat mitochondria from wildtype and UCP1 KO mice, comparing the FCCP and DNP effects with those of a fatty acid (oleate), a bona fide activator of UCP1. Whereas the sensitivity of brown-fat mitochondria to oleate was much higher in UCP1-containing than in UCP1 KO mitochondria, there was no difference in sensitivity to FCCP and DNP between these mitochondria, neither in oxygen consumption rate nor in membrane potential studies. Correspondingly, the UCP1-dependent ability of GDP to competitively inhibit activation by oleate was not seen with FCCP and DNP. It would thus be premature to abandon the established bioenergetic interpretation of chemical uncoupler effects in classical isolated brown-fat mitochondria—and probably also generally in this type of mitochondrial study. Understanding the molecular and structural reasons for the different outcomes of mitoplast and mitochondrial studies is a challenging task. •In patch-clamp experiments in mitoplasts, FCCP and DNP uncouple by activating UCP1.•Whether UCP1 activation is also the mechanism in intact brown-fat mitochondria has been studied.•The presence or absence of UCP1 does not affect FCCP and DNP efficiency and potency.•FCCP and DNP uncoupling is not competitive with GDP.•Thus, the uncoupling mechanism seen in mitoplasts is not directly transferable to mitochondria.