Valdivieso-Rivera, F.; Furino, V. O.; Leher, C. E.; Zanesco, A. M.; Cruz, M. K.; Gan, F. C.; Santoro, A. L.; Regina-Ferreira, L.; Santos, G. L.; Goncalves, T. T.; Leiria, L. O.; Moraes-Vieira, P. M.; Castilho, R. F.; Mori, M. A.; Velloso, L. A.; Sponton, C. H.

Brown adipose tissue (BAT) counteracts obesity-related metabolic dysfunction through both thermogenic and non-thermogenic means. However, substantial evidence indicates that obesity negatively affects BAT mitochondrial morphology and oxidative capacity, impairing systemic energy homeostasis. Motivated by this apparent contradiction, we investigated the relationship between obesity and mitochondrial dynamics, as the underlying mechanisms remain incompletely understood. Here, we identified E4BP4 as a transcriptional repressor that prevents obesity-induced mitochondrial fragmentation and oxidative dysfunction by inhibiting ceramide synthesis in brown fat. Specifically, E4BP4 interacts with PRDM16 to repress Cers6 mRNA expression and consequently reduces C16:0 ceramide levels by binding to a 65 kb upstream enhancer region of the Cers6 gene. Notably, the preservation of mitochondrial integrity in BAT by E4BP4 gain-of-function improves systemic glucose homeostasis, independent of weight loss. Collectively, our findings establish E4BP4 as a molecular safeguard against obesity-induced mitochondrial fragmentation and oxidative dysfunction, primarily by suppressing ceramide synthesis in brown fat.