Agradecimentos: We thank Dr. Elzira Saviani, Silvia Weidner, and Thomas Pitsch for their excellent technical assistance. H.S.B., S. C. C. Z., F. V., V. F., R. F. C., and M.A.M. were supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (2019/21852-1, 2022/00358-1, 2023/07882-0, 2021/08354-2, 2013/07607-8, 2017/17728-8, 2023/00229-0, 2022/13145-6, 2020/14725-0). M.A.M. was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (310287/2018- 9 and 306193/2022-1) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) (88881.143924/2017-01). A.B. was supported by the Deutsche Forschungsgemeinschaft (SFB1123-B10 & SPP2306 BA4925/2-1), the Deutsches Zentrum für Herz-Kreislauf-Forschung DZHK, and the ERC Starting Grant PROTEOFIT. The graphical abstract and cartoons in Figures 1 and 5 were created using BioRender.com Ver menos

Abstract: While mechanisms controlling mitochondrial uncoupling protein-1 (UCP1) expression and function in thermogenic adipocytes play a pivotal role in non-shivering thermogenesis (NST), it remains unclear whether F1Fo-ATP synthase function is regulated during NST. Here, we show that Inhibitory Factor 1 (IF1, encoded by Atp5if1), an inhibitor of ATP synthase hydrolytic activity, is a critical negative regulator of brown adipocyte energy metabolism. In mice, IF1 protein content is markedly diminished in brown adipose tissue (BAT) after 5 days of cold exposure. Additionally, the capacity of ATP synthase to generate mitochondrial membrane potential through ATP hydrolysis (the so-called "reverse mode" of ATP synthase) is higher in mitochondria isolated from cold- adapted mice compared to mice housed at room temperature. In vitro, IF1 overexpression results in an inability of mitochondria to sustain mitochondrial membrane potential upon adrenergic stimulation and this occurs in a UCP1-dependent manner. In brown adipocytes, IF1 silencing is sufficient to increase mitochondrial lipid oxidation and the cellular dependency on glycolysis to produce ATP. Conversely, IF1 overexpression blunts mitochondrial respiration without causing cellular energetic stress, leading to a quiescent-like phenotype in brown adipocytes. In mice, adeno-associated virus- mediated IF1 overexpression in BAT suppresses adrenergic-stimulated thermogenesis and decreases mitochondrial respiration in this tissue. Taken together, our data shows that the downregulation of IF1 upon cold serves to facilitate the reverse mode of ATP synthase to enable energetic adaptation and effectively support NST in BAT Ver menos