Adipocyte reporter assays: Application for identification of anti-inflammatory and antioxidant properties of mangosteen xanthones

Scope: Three fluorescence biosensors were developed based on a 3T3-L1 preadipocyte line that stably expressed Nfkb-RE/GFP, Fabp4-P/CFP, and Nrf2-P/YFP fluorescent reporters. We hypothesized that nutraceuticals' inflammatory, adipogenic, and antioxidant status will be identified based on the change in fluorescence in reporter adipocytes. We validated these assays with activators of NF kappa B, FABP4-regulating peroxisome proliferator activated receptor gamma, NFR2 and, thereafter, tested known and unknown properties of mangostines (MGs), the xanthone metabolites in mangosteen fruit. Methods and results: We validated inflammatory and adipogenic properties of alpha-MG using an Nfkb-RE/GFP biosensor assay. Next, we identified unique properties of gamma-MG, a minor MG xanthone. gamma-MG suppressed adipogenesis and expression of adiponectin, but inhibited the Nfkb-RE/GFP reporter and secretion of inflammatory monocyte chemotactic protein 1 as compared to the control adipocytes. We found that the inhibition of adipogenesis and Nf kappa b-mediated inflammation depends on a dose-dependent reduction of Nrf2 promoter activity by alpha-MG. The Nrf2 inhibition resulted in the reduced Pparg expression. alpha-MG did not directly influence Pparg activity in Fabp4-P/CFP adipocytes. Conclusion: alpha-MG-mediated antioxidant response via Nrf2 is a mechanism preventing adipogenesis and inflammation in adipocytes. Combined application of high-throughput biosensors could provide an effective platform for the identification of nutraceuticals and the mechanism of their actions in adipocytes and, potentially, in obese patients.