Ei24 deficiency in brown adipocytes induces severe hypothermia under cold stress independent of UCP1 activity.

Brown adipocytes facilitate non-shivering thermogenesis, which is critical for maintaining energy balance and heat production in response to environmental stimuli. Here, we delineate the physiological and biochemical role of etoposide-induced 2.4 (Ei24) in adenosine triphosphate (ATP) production and thermogenesis in brown adipocytes. We generated Ei24 adipocyte-specific knockout (EiaKO) mice that exhibited brown adipose tissue hypertrophy, lipid accumulation, and various mitochondrial abnormalities. Despite mitochondrial defects, uncoupling protein 1 (UCP1) expression and activity remained unchanged. However, those impairments caused lethal hypothermia in mice subjected to cold challenge, underscoring the key role of Ei24 in mitochondrial functions. Mechanistically, Ei24 deficiency disrupted cristae structure, dissipated mitochondrial membrane potential, and reduced matrix pH, leading to severe ATP depletion. We further identify the C-terminal region of Ei24 as essential for supporting ATP synthase function. Those bioenergetic defects not only destabilized the mitochondrial environment necessary for efficient UCP1-mediated thermogenesis, but also impaired ATP-dependent futile cycles such as SERCA-mediated calcium cycling and creatine substrate cycling. Together, our findings indicate that Ei24 functions as a thermogenic regulator that ensures mitochondrial ATP synthesis and structural integrity, enabling both coupled and uncoupled respiration in brown adipose tissue.