Citrus aurantium L. Dry Extracts Ameliorate Adipocyte Differentiation of 3T3-L1 Cells Exposed to TNFα by Down-Regulating miR-155 Expression

Abstract

Citrus aurantium L. dry extracts (CAde) improve adipogenesis in vitro. These effects are dependent from an early modulation of CCAAT/enhancer-binding protein beta (C/Ebpβ) expression and cyclic Adenosine Monophosphate (cAMP) response element-binding protein (CREB) activation. C/Ebpβ and Creb are also targets of miR-155. This study investigated whether CAde regulates miR-155 expression in the early stages of adipogenesis and whether it ameliorates adipocyte differentiation of cells exposed to tumor necrosis factor-alpha (TNFα). Adipogenic stimuli (AS) were performed in 3T3-L1 pre-adipocytes treated with CAde, TNFα, or both. Gene and miRNA expression were determined by quantitative real-time PCR. Adipogenesis was evaluated by Oil-Red O staining. CAde treatment enhanced AS effects during the early adipogenesis phases by further down-regulating miR-155 expression and increasing both C/Ebpβ and Creb mRNA and protein levels. At variance, TNFα inhibited 3T3-L1 adipogenesis and abolished AS effects on miR-155, C/Ebpβ, and Creb expression. However, in cells exposed to TNFα, CAde improved adipocyte differentiation and restored the AS effects on miRNA and gene expression at early time points. In conclusion, this study identified miR-155 down-regulation as part of the mechanism through which CAde enhances adipogenesis of pre-adipocytes in vitro. Furthermore, it provides evidence of CAde efficacy against TNFα negative effects on adipogenesis.