Bilobalide, the only sesquiterpene compound from Ginkgo biloba leaf, exhibits various beneficial pharmaceutical activities, such as antioxidant, anti-inflammation, and protective effects for the central nervous system. Several bioactive components extracted from Ginkgo biloba extract reportedly have the potential to attenuate lipid metabolism. However, the effect of bilobalide on lipid metabolism remains unclear. In this study, we used 3T3-L1 cells as the cell model to investigate the effect of bilobalide on adipogenesis. The results showed that bilobalide inhibited 3T3-L1 preadipocyte differentiation and intracellular lipid accumulation. Quantitative real-time PCR and western blotting results indicated that several specific adipogenic transcription factors and a few important adipogenesis-related genes were significantly down regulated on both mRNA and protein levels in bilobalide treatment groups. By contrast, the expression of some lipolytic genes, such as adipose triglyceride lipase, hormone-sensitive lipase (HSL), and carnitine palmitoyltransferase-1α, were all up-regulated by bilobalide treatment, and the phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase 1, and HSL were stimulated. Furthermore, bilobalide treatment partially restored AMPK activity following its blockade by compound C (dorsomorphin). These results suggested that bilobalide inhibited adipogenesis and promoted lipolysis in 3T3-L1 cells by activating the AMPK signaling pathway.

Bilobalide exhibits numerous beneficial bioactivities, including neuroprotective, anti-inflammatory, and antioxidant activity. Our previous study demonstrated that bilobalide inhibits adipogenesis and promotes lipolysis. The dose-dependent cytotoxicity was found to be specific to the mature adipocytes only, indicating the potential for regulating apoptosis in them. Herein, we aimed to investigate the apoptotic effects of bilobalide on 3T3-L1 mature adipocytes and elucidate the underlying mechanisms thereof. Flow cytometry analysis (FACS) revealed the pro-apoptotic effects of bilobalide on these cells. Bilobalide induced early apoptosis by reducing the mitochondrial membrane potential (MMP). DNA fragmentation was confirmed using TUNEL staining. Additionally, bilobalide increased the intracellular reactive oxygen species (ROS) levels and activities of Caspases 3/9. Pre-treatment with NAC (an ROS scavenger) confirmed the role of ROS in inducing apoptosis. Moreover, bilobalide up- and down-regulated the expression of Bax and Bcl-2, respectively, at the mRNA and protein expression levels; upregulated the Bax/Bcl-2 ratio; triggered the release of cytochrome c from the mitochondria; and increased the protein expression of cleaved Caspase 3, cleaved Caspase 9, and PARP cleavage. These results support the conclusion that bilobalide induces apoptosis in mature 3T3-L1 adipocytes through the ROS-mediated mitochondrial pathway, and offers potential novel treatment for obesity.