Complementary USP25 and USP28 proteostatic nodes sustain adipogenic transcriptional networks and metabolic dysfunction

Metabolic disorders are driven by dysregulated adipogenesis and lipid metabolism; however, the proteostatic mechanisms that sustain adipogenic transcriptional networks under obesogenic conditions remain poorly understood. Emerging evidence suggests that deubiquitinases (DUBs) regulate metabolic homeostasis through substrate-specific control of protein stability, raising the possibility that adipogenic programs are maintained by multiple DUB-dependent proteostatic nodes rather than by individual transcriptional regulators. Here, using AZ1 as a pharmacological tool for dual USP25/USP28 inhibition, we investigated the role of DUB-regulated proteostasis in adipocyte differentiation and metabolic dysfunction. USP25 and USP28 were concurrently upregulated in adipose tissue from obese human cohorts and high-fat diet (HFD)-fed mice. Genetic depletion of either USP25 or USP28 reduced lipid accumulation and adipogenic gene expression, whereas combined suppression produced a substantially stronger inhibitory effect, indicating that these DUBs exert complementary functions within adipogenic regulatory networks. Mechanistically, USP28 stabilized PPARγ, whereas USP25 directly interacted with and stabilized C/EBPα and C/EBPβ through catalytic deubiquitination. This activity was abolished by the catalytic mutant USP25 C178S, identifying USP25 as a previously unrecognized proteostatic regulator of the C/EBP adipogenic axis. Consistent with these findings, pharmacological inhibition of USP25/USP28 suppressed adipogenesis and lipid accumulation in vitro. In vivo, AZ1 attenuated body-weight gain and adipose tissue expansion in HFD-fed mice, improved glucose tolerance and insulin sensitivity, and reduced adipogenic regulators in adipose tissue. AZ1 also decreased hepatic triglyceride and free fatty acid accumulation while increasing hepatic PPARα expression, supporting systemic metabolic improvement associated with altered adipose–liver crosstalk. Collectively, our findings identify USP25–C/EBPα/β and USP28–PPARγ as complementary DUB-dependent proteostatic nodes that cooperatively sustain adipogenic transcriptional networks. These results support a model in which pathological adipogenesis is maintained through coordinated proteostatic regulation of multiple transcriptional axes rather than through a single dominant adipogenic regulator and suggest that disruption of complementary DUB-regulated pathways may represent an effective strategy for metabolic disease intervention.