Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant V phenolic compound with apoptotic activity in cancer cells, on 3T3-L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3-L1 pre-confluent preadipocytes and lipid-filled adipocytes were incubated with esculetin (0 to 800 mu M) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single-stranded DNA. Post-confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red 0 for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time- and dose-related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 mu M esculetin (p < 0.05), and after 48 hours, as little as 50 mu M esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 mu M esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time- and dose-related manner, beginning as early as 6 hours with 400 and 800 mu M esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3-L1 preadipocytes. Esculetin-mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3-L1 cells.