Agnuside Stabilizes the Complex I Assembly Factor NDUFAF6 to Reinforce Mitochondrial Efficiency and Thermogenic Responsiveness

ABSTRACT Brown and beige adipocytes dissipate energy as heat, yet effective strategies to enhance their mitochondrial efficiency remain limited. Here, we identify Agnuside (AGN) as a selective stabilizer of the complex I assembly factor NDUFAF6. AGN directly binds cytosolic NDUFAF6, suppresses its ubiquitination, prolongs its half‐life, and facilitates mitochondrial import, thereby reinforcing complex I assembly and promoting coordinated stabilization of complexes III and IV within the respirasome, without altering complex II, complex V, or global mitochondrial biogenesis. Functionally, AGN exhibits a demand‐dependent metabolic profile. Under basal conditions, AGN enhances mitochondrial oxidative efficiency without activating overt UCP1‐dependent uncoupling. In contrast, cold exposure or chronic high‐fat feeding markedly potentiates its thermogenic impact, as evidenced by improved mitochondrial ultrastructure, increased UCP1 abundance, and elevated energy expenditure in brown adipose tissue, with similar mitochondrial reinforcement observed in inguinal white adipose tissue under sustained metabolic stress. Importantly, thermoneutral Ucp1 knockdown does not abolish AGN‐mediated enhancement of respiratory complex assembly and ATP production, whereas genetic ablation of Ndufaf6 eliminates these effects. Together, these findings establish AGN‐NDUFAF6 stabilization as a key regulatory mechanism governing adipose mitochondrial efficiency and thermogenic responsiveness, and highlight assembly‐factor targeting as a promising strategy to restore oxidative metabolism in metabolic dysfunction.