Liraglutide Attenuates Aβ42 Generation in APPswe/SH-SY5Y Cells Through the Regulation of Autophagy

Abstract

Objective: This study aimed to clarify whether liraglutide, a GLP-1 analogue, can ameliorate Aβ pathology through the regulation of autophagy in Alzheimer's disease (AD) and to explore the related mechanisms thereof.

Methods: We used SH-SY5Y cells transiently transfected with APP695swe plasmid as an AD cellular model. Transfected cells were treated with liraglutide for 24 h in the presence or absence of 3-MA. Autophagy markers and the Aβ level were then evaluated by Western blot and ELISA. We also investigated the potential involvement of mTOR and JNK pathway in liraglutide-mediated autophagy.

Results: Our results showed that liraglutide reduced Aβ42 generation and enhanced autophagy in APPswe/SH-SY5Y cells; however, these effects could be counteracted with 3-MA. Furthermore, our data showed that liraglutide-induced autophagy does not follow the mTOR pathway. Liraglutide might promote autophagy in APPswe/SH-SY5Y cells by activating the JNK pathway and inhibiting the beclin-1/bcl-2 complex.

Conclusion: Here, we report a novel mechanism underlying liraglutide-attenuated Aβ42 generation through the activation of autophagy in AD cellular model.

Keywords: Alzheimer’s disease; Aβ; JNK; autophagy; glucagonlike peptide 1.

Figure 1

The effects of liraglutide and rapamycin on autophagy markers in APPswe/SH-SY5Y cells. (A–D) Western blotting data show that liraglutide significantly upregulates the LC3II/LC3I ratio and the level of beclin-1 protein, but down-regulates the level of p62 protein in APPswe/SH-SY5Y cells, compared to APP control. These results are similar to the effects of Rapamycin on autophagy in APPswe/SH-SY5Y cells. Data were presented as mean ± SD from three independent experiments. Data were analysed by one-way ANOVA followed by Student Newman–Keuls test. *p<0.05 vs APP control, **p<0.01 vs APP control.

Figure 2

3-MA blocks the effect of liraglutide on autophagy in APPswe/SH-SY5Y cells. (A–D) Western blotting data show that the autophagy inhibitor 3-MA significantly blocks the liraglutide-upregulated LC3II/LC3I ratio and protein level of beclin-1, as well as the liraglutide-downregulated expression of the p62 protein in APPswe/SH-SY5Y cells. Data were presented as mean ± SD from three independent experiments. Data were analysed by one-way ANOVA followed by Student Newman–Keuls test. *p<0.05 vs APP control,**p<0.01 vs APP control.

Figure 3

The effect of liraglutide on the generation of Aβ₄₂ in APPswe/SH-SY5Y cells. ELISA results show that liraglutide significantly reduces the level of Aβ₄₂ in the culture medium of APPswe/SH-SY5Y cells, compared to APP control. The decrease of extracellular Aβ₄₂ induced by liraglutide can be blocked by 3-MA and SP600125. Data were presented as mean ± SD from three independent experiments. Data were analysed by one-way ANOVA followed by Student Newman–Keuls test. ^##p<0.01 vs NC group, *p<0.05 vs APP control.

Figure 4

The PI3K/AKT/mTOR signaling pathway is dispensable for liraglutide-induced autophagy. (A) Western blotting data show that liraglutide significantly upregulates p-AKT/AKT ratio in APPswe/SH-SY5Y cells, compared to APP control. LY294002 blocks the liraglutide-induced phosphorylation of AKT. (B) Western blotting data show that liraglutide significantly upregulates p-mTOR/mTOR ratio in APPswe/SH-SY5Y cells, as compared to APP control. Rapamycin blocks the liraglutide-induced phosphorylation of mTOR. Data were presented as mean ± SD from three independent experiments. Data were analysed by one-way ANOVA followed by Student Newman–Keuls test. **p<0.01 vs APP control.

Figure 5

Induction of autophagy by liraglutide through activating the JNK signaling pathway. (A–E) Western blotting results show that liraglutide significantly increases the ratio of p-JNK/JNK in APPswe/SH-SY5Y cells compared to APP control. The liraglutide-induced phosphorylation of JNK and the increased LC3II/LC3I ratio can be blocked by SP600125. Furthermore, liraglutide significantly reduces bcl-2 expression, but increases beclin-1 expression, the effect of which can be blocked by SP600125. Data were presented as mean ± SD from three independent experiments. Data were analysed by one-way ANOVA followed by Student Newman–Keuls test. *p<0.05 vs APP control, **p<0.01 vs APP control, ^##p<0.01 vs liraglutide group.