Anti-adipogenic effect and mechanism in 3T3-L1 preadipocytes by cyclosporin A, an immunosuppressant

Abstract

Adipogenesis, also called the differentiation of preadipocytes, leads to the phenotype of mature adipocytes. However, excessive adipogenesis is closely linked to the development of obesity. Cyclosporin A, a neutral lipophilic cyclic undecapeptide isolated from the fungus Hypocladium inflatum gams, is known for several biological activities, including immunosuppression and anti-inflammation. Little is known about the relationship between cyclosporin A and obesity. In this study, we investigated the effect of cyclosporin A on adipogenesis in 3T3-L1 murine preadipocytes. 3T3-L1 preadipocyte differentiation was evaluated by Oil Red O staining and AdipoRed assay. The expression and phosphorylation levels of adipogenesis-related proteins in differentiating 3T3-L1 cells were evaluated by Western blotting. The mRNA expression levels of adipogenesis-related proteins in differentiating 3T3-L1 cells were measured by RT-PCR. Cyclosporin A considerably inhibited lipid accumulation and reduced triglyceride (TG) contents during the differentiation of 3T3-L1 preadipocytes into adipocytes in a concentration-dependent manner, suggesting its anti-adipogenic effect. Treatment with cyclosporin A at the concentrations tested was not cytotoxic to 3T3-L1 cells. Mechanistically, at a 10 μM concentration, cyclosporin A strongly down-regulated the protein and mRNA expressions of two adipogenic transcription factors, CCAAT/enhancer-binding protein-α (C/EBP-α) and peroxisome proliferator-activated receptor-γ (PPAR-γ) in differentiating 3T3-L1 cells. Furthermore, at a 10 μM concentration, cyclosporin A reduced the phosphorylation levels of signal transducers and activator of transcription-3 (STAT-3), other adipogenic transcription factor, and suppressed the protein and mRNA expressions of perilipin A, a protein that binds to and stabilizes lipid droplets, in differentiating 3T3-L1 cells. However, treatment with 10 μM of cyclosporin A did not modulate the protein and mRNA expressions of fatty acid synthase during 3T3-L1 preadipocyte differentiation. Collectively, these results show that cyclosporin A inhibits adipogenesis in differentiating 3T3-L1 cells, and that this inhibition is mediated through the reduced expression and phosphorylation levels of C/EBP-α, PPAR-γ, perilipin A, and STAT-3.