Irisin Exerts Inhibitory Effect on Adipogenesis Through Regulation of Wnt Signaling

Abstract

Irisin is an exercise-induced myokine known to induce adipocyte browning through induction of uncoupling protein 1. Recent studies have reported that irisin is also an adipokine. However, there is limiting evidence on the role of endogenous irisin from adipocytes. In this study we aim to elucidate the expression and secretion pattern of irisin during adipocyte differentiation and the role of endogenous and exogenous irisin on the adipogenic process. As such, recombinant irisin, plasmid expressing FNDC5 and small interfering RNA were utilized. Our results show that the gene expression of irisin precursor FNDC5 and irisin secretion increases at the early stage of adipogenesis. Both recombinant irisin treated cells and FNDC5-overexpressed cells resulted in inhibition of adipogenesis evidenced by downregulated C/EBPα, PPARγ, and FABP4 expression and reduced lipid accumulation. Further data showed that the inhibitory effect of irisin on adipogenesis is mediated though potentiation of Wnt expression, which is known to determine the fate of mesenchymal stem cells and regulate adipogenesis. Conversely, FNDC5 knockdown cells showed downregulated Wnt expression, but failed to further induce adipogenesis. This study suggests that both exogenous and endogenous irisin is able to inhibit adipogenesis and that activation of Wnt and subsequent repression of transcription factors is partly involved in this process. This provides a novel insight on the local effect of irisin on adipocytes and additional benefit to protect against obesity-related metabolic disorders.

Keywords: FNDC5; Wnt; adipogenesis; irisin; myokine.

FIGURE 1

Irisin gene expression and secretion during 3T3-L1 adipogenesis. (A,B) Gene expressions of PPARγ and FNDC5 were examined by real-time PCR during day D–2 to D+8 of adipogenesis. Quantifications were normalized to 18S in each reaction. ^∗P < 0.05 compared with D–2, †P < 0.05 compared with D–0. (C) Irisin secretion in media during adipogenesis was analyzed by ELISA. ^∗P < 0.05 compared with D–0.

FIGURE 2

Effect of recombinant irisin and FNDC5 overexpression on adipogenesis. (A–C) 3T3-L1 cells were treated with or without 100 ng/ml irisin from D–2 to D+6 of adipogenesis. Gene expressions of transcription factors were measured by real-time PCR and normalized to 18S rRNA. (D–J) 3T3-L1 preadipocytes were transfected with control or plasmid expressing FNDC5. Two days confluent cells were induced to differentiate and were harvested at day + 5 and day + 8. (D–F) Gene expressions of PPARγ, CEBPα, and FABP4 were analyzed by real-time PCR and normalized to 18S. (G,H) Western blot analysis of transcription factors. (I,J) Representative pictures and quantification of Oil red O staining in FNDC5-overexpressed cells at day 8. ^∗P < 0.05 compared with control at each time-point.

FIGURE 3

Effect of recombinant irisin treatment on Wnt expression. (A–C) Gene expression analysis of Wnt ligands during 3T3-L1 adipocyte differentiation by real-time PCR. Quantification were normalized to 18S rRNA ^∗P < 0.05 compared with D–0. (D–F) Gene expression of analysis of Wnt ligands at early stage of adipogenesis by real-time PCR. ^∗P < 0.05 compared with D–2, †P < 0.05 compared with D–0. (G–J) 3T3-L1 cells were treated with 100 ng/ml recombinant irisin at D–2. (G–I) Wnt gene expressions were measured by real-time PCR and normalized to 18S rRNA. (J) Protein expression of Wnt10a with or without recombinant irisin treatment were analyzed by Western blot, ^∗P < 0.05 compared with control.

FIGURE 4

Effect of FNDC5 silencing on Wnt gene expression and adipogenesis. 3T3-L1 preadipocytes were transfected with FNDC5 siRNA and negative control at 25 nmol/L for 48 h. (A–C) Cells were harvested 1 day after MDI treatment. Gene expression of Wnt ligands were examined by real-time PCR and normalized to 18S in each reaction. (D,E) Representative pictures and quantification of Oil red O staining after siFNDC5 transfection. The adipogenesis was induced by conventional MDI treatment or with addition of PPARγ agonist, rosiglitazone, and harvested on day 8. (F–H) Real-time PCR analysis of transcription factors after siFNDC5 transfection. Data is shown as ^∗P < 0.05 compared with siControl.

FIGURE 5

Schematic diagram of the effect of irisin on adipogenesis.