Liraglutide Inhibits the Apoptosis of MC3T3-E1 Cells Induced by Serum Deprivation through cAMP/PKA/β-Catenin and PI3K/AKT/GSK3β Signaling Pathways

Abstract

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of GSK3β, increased β-catenin phosphorylation at Ser675 site and upregulated nuclear β-catenin content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, β-catenin abrogated the liraglutide-induced activation of cAMP, AKT, β-catenin, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through cAMP/PKA/β-catenin and PI3K/Akt/GSK3β signaling pathways.

Keywords: apoptosis; liraglutide; osteoblast; signaling pathway.

Fig. 1. GLP-1R was expressed in MC3T3-E1 osteoblasts

RT-PCR (A), Western blot (B), and confocal microscopy (C) indicating the presence of the GLP-1 receptor in MC3T3-E1 cells and mouse pancreas (as positive control). (C) 400×magnification. The results were normalized to GAPDH as an internal control. Experiments were performed at least three times.

Fig. 2. Effect of liraglutide on serum deprivation-induced apoptosis in osteoblastic MC3T3-E1 cells. Cells were deprived of serum for 24 h, then exposed to liraglutide (0, 10, 100, or 1000 nM) for 48 h

(A) The nucleus of the MC3T3-E1 cells were dyed with Hoechst 33258 and representative images of Hoechst 33258-stained cells were obtained under a fluorescence microscope. (B) Apoptosis was determined by annexin V-propidiumiodide (PI) double staining. (C) The percentage of apoptotic cells by flow cytometry was lower for cells treated with liraglutide than control group (*P < 0.05, **P < 0.01). (D) Cell death detection ELISA showed that liraglutide suppressed the apoptosis of MC3T3-E1 cells. (E) The expression of apoptosis related proteins Bcl-2, Bax, caspase-3 were determined by Western blot. (F) The Bcl-2/Bax ratio was calculated. Data are shown as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 compared with the control group.

Fig. 3. Activation of cAMP/PKA/β-catenin and PI3K/AKT/GSK3β/β-catenin signaling pathway in liraglutide-stimulated osteoblastic MC3T3-E1 cells

Cells were exposed to 100 nM liraglutide and 20 μM H89 or 20 μM LY294002, respectively. Cell lysates were subjected to Western blotting and ELISA to assess AKT (A, B), GSK3β (A, C), β-catenin (D, E), cAMP (F) activation. β-catenin level in the cytoplasm and nucleus (G–I) and TCF7L2 mRNA expression (J) were detected to evaluate the canonical Wnt/β-catenin signaling pathway activation.

Fig. 4. Liraglutide-induced signaling pathways through GLP-1R were regulated by β-catenin

Cells were treated with GLP-1R, or β-catenin siRNA in the presence of 100 nM liraglutide. The transfection efficiency was determined by Western blot (A, B). The intracellular levels of cAMP (C), AKT (D, E) and β-catenin (D, F) activation were examined.

Fig. 5. Effect of specific selective inhibitors and siRNAs on liraglutide-induced decrease of osteoblastic MC3T3–E1 cells apoptosis

(A) Cells were pretreated with 20 μM H89 or 20 μM LY294002, respectively, for 2 h before incubation with 100 nM liraglutide for 48 h. (B) Cells were also treated with the GLP-1R or β-catenin-siRNA in the presence of 100 nM liraglutide for 48 h. Cell death detection ELISA was performed to measure MC3T3-E1 cells apoptosis. Data are shown as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 compared with the control group.

Fig. 6. Model for liraglutide-mediated anti-apoptotic effect via GLP-1R in osteoblast