Shabalina Irina G, Jiménez Beatriz, Sousa-Filho Celso Pereira Batista, Cannon Barbara, Nedergaard Jan
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91, Sweden.
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91, Sweden.
Biochim Biophys Acta Bioenerg. 2025 Jan 1;1866(1):149516. doi: 10.1016/j.bbabio.2024.149516. Epub 2024 Sep 30.
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.
最近对线粒体膜间腔的膜片钳研究对传统观点提出了挑战，即经典化学解偶联剂（如FCCP或DNP）的作用是由于这些物质本身具有质子载体特性。相反，这些研究表明，在棕色脂肪线粒体中，FCCP和DNP诱导的解偶联是通过UCP1的激活介导的（在其他组织中是通过腺嘌呤核苷酸转运体的激活）。因此，这些研究倡导了一种全新的范式来解释标准生物能量学实验。为了检验在棕色脂肪线粒体膜间腔中获得的这些膜片钳结果是否能直接应用于经典的分离棕色脂肪线粒体研究，我们研究了FCCP和DNP对野生型和UCP1基因敲除小鼠棕色脂肪线粒体的影响，并将FCCP和DNP的作用与UCP1的真正激活剂脂肪酸（油酸）的作用进行了比较。虽然含UCP1的棕色脂肪线粒体对油酸的敏感性远高于UCP1基因敲除的线粒体，但这些线粒体对FCCP和DNP的敏感性在耗氧率和膜电位研究中均无差异。相应地，FCCP和DNP不存在GDP依赖的竞争性抑制油酸激活的能力。因此，放弃对经典分离棕色脂肪线粒体中化学解偶联剂作用的既定生物能量学解释可能为时过早，而且在这类线粒体研究中可能普遍如此。理解线粒体膜间腔和线粒体研究结果不同的分子和结构原因是一项具有挑战性的任务。
