fsnRNA-seq Reveals Subcutaneous White Adipose Tissue Remodeling Upon Return to Thermoneutrality After Cold Stimulation

Yusha Yang^1,2Guanyu Zhang²Ting Yi^1,2Shuran Yang^2,3Shuai Wu²Yongqiang Zhang²Li Zhang²Li Xi²Xiuxuan Wu^1,2Jun Li^1*Danfeng Yang^2*

• ¹Guizhou Medical University, Guiyang, China
• ²Academy of Military Medical Sciences (AMMS), Beijing, Beijing Municipality, China
• ³Tianjin University of Traditional Chinese Medicine, Tianjin, China

Cold stimulation induces browning of subcutaneous white adipose tissue (sWAT), making it a prime target for treating obesity and metabolic disorders. However, this remodeling is reversible: upon return to thermoneutrality (rewarming), sWAT whitens and loses its enhanced metabolic functions. Given the limited understanding of the microscopic dynamic changes and underlying mechanisms during this process, we established a temporally dynamic mouse model spanning the entire period from cold stimulation to the return to thermoneutrality, with inguinal sWAT (iWAT) selected as the study subject. Based on preliminary data demonstrating stabilization in iWAT histology, expression levels of key thermogenic proteins, and the bulk transcriptome, we selected the two-week time point after the return to thermoneutrality for detailed analysis. Subsequently, we employed single-nucleus RNA sequencing (snRNA-seq) to comprehensively characterize iWAT cellular dynamics during cold stimulation and the subsequent two-week period after the return to thermoneutrality. Our findings revealed that while iWAT phenotypically reverts to a white state after 2 weeks of rewarming, as evidenced by structural, functional, and bulk transcriptomic characteristics, significant cold-induced molecular and cellular signatures persist. Specifically, we observed altered differentiation trajectories in both adipose stem and progenitor cells (ASPCs) and adipocytes, suggesting dedifferentiation and reprogramming tendencies. Furthermore, the ANGPTL signaling pathway, activated in thermogenic adipocyte subpopulation A3 during cold stimulation, remained active and influenced cell-cell communication even after the loss of thermogenic capacity. These findings provide novel insights into the complex cellular and molecular mechanisms governing iWAT plasticity and suggest a potential role for ANGPTL signaling in maintaining the white phenotype following cold stimulation.