Enhanced effects of 1,25(OH)2D3 plus genistein on adipogenesis and apoptosis in 3T3-L1 adipocytes

objective: To investigate the ability of 1,25(OH) 2 D 3 (D) and genistein (G), alone and in combination, to inhibit adipogenesis and induce apoptosis in 3T3-L1 adipocytes. Methods and Procedures: 3T3-L1 preadipocytes and mature adipocytes were incubated with various concentrations of D and G, alone and in combination, for 48 h. Viability was determined using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay. Post-confluent preadipocytes were incubated with D and G for up to 6 days during adipogenesis and lipid content was quantified by Nile Red dye; apoptosis was quantified by measurement of single-stranded DNA. Expression of adipocyte-specific proteins and VDR was analyzed by western blotting. Results: Combining D and G did not cause an enhanced effect on cell viability in either preadipocytes or mature adipocytes. In maturing preadipocytes, D at 0.5 nmol/l (D0.5) increased apoptosis by 47 +/- 10.25% (P < 0.05) and inhibited lipid accumulation by 28 +/- 10% (P < 0.001), while G at 25 mu mol/l (G25) had no significant effect. However, D+G caused an enhanced apoptosis by 136 +/- 12.6% (P < 0.001) and enhanced inhibition of lipid accumulation by 82.46 +/- 2.95% (P < 0.001). Similarly, D0.5 alone decreased adipose-specific gene 422 (aP2) expression to 34.2 +/- 2.3% and increased VDR expression levels by 41.8 +/- 11% (P < 0.001), but G25 showed no effect. However, D0.5+G25 decreased aP2 expression to 52 +/- 4.2% (P < 0.05) and increased VDR expression levels by 131 +/- 14.5% (P < 0.0001). Discussion: These findings suggest that combining 1,25(OH) 2 D 3 with genistein results in an enhanced inhibition of lipid accumulation and induction of apoptosis in maturing 3T3-L1 preadipocytes.