UCP1 neddylation regulates white adipocyte browning through NIX-mediated mitophagy

Abstract Background Adipocyte browning contributes to energy homeostasis and represents a promising strategy for obesity treatment. Uncoupling protein 1 (UCP1), a well-established marker of beige adipocytes, has been regarded as a key effector that promotes energy expenditure and improves glucose metabolism. However, the observations that Ucp1 -knockout ( Ucp1 −/− ) mice exhibit unexpected resistance to obesity and increased beige adipocyte-like features in white adipose tissue (WAT) have rendered the precise role of UCP1 in browning controversial. We investigated the role of UCP1 neddylation in white adipocytes and its relevance in high-fat diet (HFD)-induced obesity. Methods The impact of neddylation inhibition on 3T3-L1 cells during white adipocyte differentiation was assessed by evaluating lipid accumulation, browning markers, and mitochondrial function. UCP1 neddylation and its downstream regulation of ubiquitination and mitophagy were examined with co-immunoprecipitation and transmission electron microscopy (TEM). Site-specific UCP1 lysine mutants and murine double minute 2 ( MDM2 ) knockdown models were further used to identify the neddylation sites and the responsible E3 ligase. Functional validation was conducted in HFD-induced obese Ucp1 +/+ and Ucp1 −/− mice. Each genotype was randomized to receive either MLN4924 or vehicle. Assessments included histological evaluation, glucose and insulin tolerance tests, and metabolic analysis using the Comprehensive Lab Animal Monitoring System (CLAMS). Results Neddylation of UCP1 at K138 by MDM2 facilitates NIX-mediated mitophagy through the interaction between UCP1 and NIX. At a different site, neddylation of UCP1 at K237 by MDM2 promotes UCP1 proteasomal degradation. Blockade of neddylation induces beige adipocyte features in white adipocytes, characterized by UCP1 accumulation and increased mitochondrial content. Consistently, MLN4924 treatment reduced body weight, enhanced thermogenic capacity, and improved energy metabolism in Ucp1 +/+ mice. These treatment effects were not observed in Ucp1 −/− mice, indicating a UCP1-dependent pathway. Conclusions This study identifies UCP1 neddylation as a dual regulatory mechanism controlling UCP1 turnover and mitophagy in white adipocytes. These findings provide a post-translational explanation for the paradoxical metabolic phenotypes associated with altered UCP1. Targeting UCP1 neddylation promotes adipocyte browning and improves energy metabolism, representing a potential therapeutic strategy for obesity.