Irisin alleviates obesity-induced bone loss by inhibiting interleukin 6 expression via TLR4/MyD88/NF-κB axis in adipocytes

Abstract

Introduction: Obesity-induced bone loss affects the life quality of patients all over the world. Irisin, one of the myokines, plays an essential role in bone and fat metabolism.

Objective: Investigate the effects of irisin on bone metabolism via adipocytes in the bone marrow microenvironment.

Methods: In this study, we fed fibronectin type III domain-containing protein 5 (FNDC5, the precursor protein of irisin) knockout mice (FNDC5^-/-) with a high-fat diet (HFD) for 10 weeks. The quality of bone mass was assessed by micro-CT analysis, histological staining, and dynamic bone formation. In vitro, the lipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was assayed by Oil Red O staining, and the osteogenic differentiation was assayed by alkaline phosphatase staining. Meanwhile, the gene expression in the BMSC-differentiated adipocytes by RNA sequence and the involved pathway of irisin were determined by western blot and qRT-PCR were performed.

Results: The FNDC5^-/- mice fed with a HFD showed an increased body weight, fat content of the bone marrow and bone, and a decreased bone formation compared with those with a standard diet (SD). In vitro, irisin inhibited the differentiation of BMSCs into adipocytes and alleviated the inhibition of osteogenesis derived from BMSCs by the adipocyte supernatant. RNA sequence and blocking experiment showed that irisin reduced the production of interleukin 6 (IL-6) in adipocytes through downregulating the TLR4/MyD88/NF-κB pathway. Immunofluorescence staining of bone marrow further confirmed an increased IL-6 expression in the FNDC5^-/- mice fed with HFD compared with those fed with SD, which suffered serious bone loss.

Conclusion: Irisin downregulates activation of the TLR4/MyD88/NF-κB pathway, thereby reducing IL-6 production in adipocytes to enhance the osteogenesis of BMSCs. Thus, the rescue of osteogenesis of BMSCs, initially inhibited by IL-6, is a potential therapeutic target to mitigate obesity-induced osteoporosis.

Keywords: Adipocytes; IL-6; Irisin; Osteogenesis; TLR4/MyD88/NF-κB.

Graphical abstract

Fig. 1

Irisin deficiency exacerbated HFD-induced obesity and fat content. (A) Gross observation of mice after 10 weeks of dietary intervention. (B) Body mass of the WT and FNDC5^-/- mice. (n = 5) (C) ORO staining and quantification of mouse femur sections. (n = 3).

Fig. 2

Irisin deficiency exacerbated HFD-induced bone mass imageologically. The femurs were scanned by Micro-CT. (A) 2D reconstruction of the femurs. (B) 3D reconstruction of the femurs. (C) The trabecular bone parameters included BV/TV, BMD, Tb. N, Tb. Th, Tb. Sp, Conn. D, and SMI. (n = 5).

Fig. 3

Irisin deficiency accelerated HFD-induced bone mass histologically. (A) H&E staining of the femur and quantification analysis. (n = 3) (B) Masson staining of the femur and quantification analysis. (n = 3).

Fig. 4

Irisin deficiency exacerbated the decrease in bone formation induced by HFD. (A) Representative images of calcein fluorescent staining. (B) RUNX2 expression in femur detected by IHC. (C) Osterix expression in femur detected by IHC. (D) The mineralized deposition rate (MAR) of bone trabeculae was quantified. (n = 3) (E) Quantitative analysis of RUNX2⁺ cells. (n = 3) (F) Quantitative analysis of Osterix ⁺ cells. (n = 3) (G) PINP in sera by ELISA. (n = 5) (H) CTX in sera by ELISA. (n = 5).

Fig. 5

Irisin inhibited BMSC-derived adipogenesis. BMSCs were induced to adipogenesis for 14 days. (A) ORO staining of the adipocytes. (B) Quantification of ORO staining of the adipocytes. (n = 3) (C) PPARγ gene expression in adipocytes by qRT-PCR. (n = 3).

Fig. 6

Irisin alleviated the inhibition of adipocytes on BMSC osteogenesis. BMSCs were cultured in the osteogenic medium with CM. (A) ALP staining after 7 days. (B) ARS staining after 14 days. (C) Quantification of ALP activity. (n = 3) (D) Quantitative of mineralized nodules. (n = 3) (E-H) Gene expression of Alp, Opn, Runx2, and Ostrix detected by qRT-PCR after 7 days. (n = 3).

Fig. 7

Irisin reduced the expression of genes related to inflammatory factors in adipocytes. BMSCs were induced to adipocytes for 14 days with or without irisin and RNA-sequence were performed. (A) Volcano plot showing differentially regulated genes. (B) Heatmap showing inflammation-related gene expression. (C) KEGG enrichment analysis.

Fig. 8

Irisin downregulated the TLR4/NF-κB pathway in adipocytes. (A) Heatmap showing TLR-related gene expression. (B) Heatmap showing NF-κB-related gene expression. (C) GO enrichment analysis of differential genes. (D) Chord diagram showing KEGG enrichment analysis.

Fig. 9

Irisin suppressed BMSC-differentiated adipocytes to secret IL-6 though the TLR4/NF-κB pathway. (A) Gene expressions of Il-6, Vcam1, Tlr4, MyD88, and NF-κB detected by qRT-PCR. (n = 3) (B) Protein expressions of IL-6, VCAM1, TLR4, MyD88, and NF-κB detected by WB. (n = 3) (C) IL-6 in the cell supernatant determined by ELISA. (n = 5).

Fig. 10

Irisin reduced cellular IL-6 production by decreasing TLR4 expression in adipocytes. (A) IF staining for IL-6 in BMSC-derived adipocytes. (B) IF staining for IL-6 and TLR4 in BMSC-derived adipocytes.

Fig. 11

Irisin regulated IL-6 production by TLR4/MyD88 signaling in adipocytes. (A) ORO staining of BMSC-differentiated adipocytes after induction for 14 days. (n = 3) (B) ALP staining and ALP activity of BMSC-differentiated osteoblasts 7 days after cultured with the osteogenic conditional medium. (n = 3) (C) ARS staining and quantification of mineralized nodules of BMSC-differentiated osteoblasts 14 days after cultured with the osteogenic conditional medium. (n = 3) (D) The gene expression of Il-6, Tlr-4, MyD88 and NF-κB detected by qRT-PCR. (n = 3) (F) The protein expression IL-6, TLR-4, MyD88 and NF-κB detected by WB. (n = 3).

Fig. 12

Irisin deficiency increased TLR4 and IL-6 expression in the bone marrow cavity of the mice. (A) IF staining of IL-6 in the femur. (B) IF staining of TLR4 in the femur.