1699-P: The Conversion and Potential Mechanism of Conditionally Immortalized Human Classical Brown Preadipocytes from Proliferative to Adipogenic States

Introduction & Objective: Considering to the anti-obesity and diabetic effect of brown adipose tissue (BAT), different immortalized brown preadipocytes cell lines have been established. Notably, murine preadipocyte cell lines expressed Simian virus 40 large tumor antigen (SV40LT) possess adipogenic capacity, whereas human preadipocyte cell lines with the same method failed. This study aimed to establish conditionally immortalized human classical brown preadipocytes (hcBPAs) and elucidate the mechanism underlying SV40LT-mediated regulation of adipogenesis. Methods: hcBPAs was achieved by controlling SV40LT with doxycycline-inducible Tet-on system. Proliferation ability, adipogenic capacity, and brown adipocytes identity were compared between hcBPAs and primary brown preadipocytes. hcBPAs were transplanted into HFD-induced nude mice. Metabolic index and adipogenic capacity were evaluated after 8 weeks. ATAC-seq and RNA-seq were adopted between doxycycline-removal and -inducible hcBPAs to elucidate changes in chromatin accessibility and adipogenic genes. Results: A hcBPAs was obtained with similar adipogenic capacity, thermogenic function, and molecular characteristics compared with primary preadipocytes. hcBPAs transplantation models have shown improvements in glucose metabolism and reconstituted functional brown adipose tissue. Differentiated doxycycline-removal hcBPAs showed increased ATAC sensitivity and mRNA expressions in adipogenic related genes and transcription factor (Fosl2 and CTCF) occupancy. RNA-seq and qPCR revealed equal expression levels of these regulatory factors in both groups, raising the possibility that downregulated of SV40TL expression promotes the binding of these factors to chromatin. Conclusion: The model of hcBPAs was constructed and provided a reliable tool for prevention and treatment of obesity. Keywords: hcBPAs, SV40TL, ATAC sensitivity, Fosl2, CTCF Disclosure L. You: None. S. Hua: None. T. Yu: None. C. Ji: None. Funding National Natural Science Foundation of China (82170880)