DPP-4 Inhibitor Linagliptin Attenuates Aβ-induced Cytotoxicity through Activation of AMPK in Neuronal Cells

Abstract

Aim: It is now clear that insulin signaling has important roles in regulation of neuronal functions in the brain. Dysregulation of brain insulin signaling has been linked to neurodegenerative disease, particularly Alzheimer's disease (AD). In this regard, there is evidence that improvement of neuronal insulin signaling has neuroprotective activity against amyloid β (Aβ)-induced neurotoxicity for patients with AD. Linagliptin is an inhibitor of dipeptidylpeptidase-4 (DPP-4), which improves impaired insulin secretion and insulin downstream signaling in the in peripheral tissues. However, whether the protective effects of linagliptin involved in Aβ-mediated neurotoxicity have not yet been investigated.

Methods: In the present study, we evaluated the mechanisms by which linagliptin protects against Aβ-induced impaired insulin signaling and cytotoxicity in cultured SK-N-MC human neuronal cells.

Results: Our results showed that Aβ impairs insulin signaling and causes cell death. However, linagliptin significantly protected against Aβ-induced cytotoxicity, and prevented the activation of glycogen synthase kinase 3β (GSK3β) and tau hyperphosphorylation by restoring insulin downstream signaling. Furthermore, linagliptin alleviated Aβ-induced mitochondrial dysfunction and intracellular ROS generation, which may be due to the activation of 5' AMP-activated protein kinase (AMPK)-Sirt1 signaling. This upregulation of Sirt1 expression was also observed in diabetic patients with AD coadministration of linagliptin.

Conclusions: Taken together, our findings suggest linagliptin can restore the impaired insulin signaling caused by Aβ in neuronal cells, suggesting DPP-4 inhibitors may have therapeutic potential for reducing Aβ-induced impairment of insulin signaling and neurotoxicity in AD pathogenesis.

Keywords: AMP-activated protein kinase; Alzheimer's disease; Amyloid-β; Linagliptin; Sirtuin 1.

Figure 1

Effects of linagliptin on cell viability and incretin‐related mRNA expression in SK‐N‐MC neuronal cells. (A) Dose effects of linagliptin on SK‐N‐MC cells by MTT assay. Linagliptin shows no significant cytotoxicity <50 μM. (B) 50 μM of linagliptin causes no significant alteration of cell viability within a 48‐h period. (C) SK‐N‐MC cells are treated with or without 50 μM of linagliptin for 24 h. The mRNA levels of incretin‐related target genes including insulin, insulin‐like growth factor‐1 (IGF‐1), and glucagon‐like peptide 1 (GLP‐1) are measured by using real‐time qPCR, and the results are presented as means ± standard error of the means (SEM) of three independent experiments. *P < 0.05 and **P < 0.01 by using multiple comparisons of Dunnett's post‐hoc test. N. S., no significant difference.

Figure 2

Linagliptin protects against Aβ‐induced SK‐N‐MC cell death. (A) MTT assays indicate 2.5 μM of Aβ markedly induces cell death after 24 h of incubation. However, linagliptin significantly prevents Aβ‐induced neurotoxic effects in a dose‐dependent manner. (B) Western blotting results demonstrate that linagliptin (50 μM) treatment suppresses both caspase 3 and PARP activation induced by Aβ (2.5 μM). (C) Linagliptin (50 μM) markedly reduces 2.5 μM of Aβ‐induced nucleus fragmentation. Apoptosis is determined by fragmented morphology in the nucleus for DAPI fluorescence. The numbers of apoptotic cells are quantified by averaging cell counts in twenty random 400× fields. Other data were performed in three independent experiments, and values are presented as mean ± SEM. Significant differences was determined by using the multiple comparisons of Dunnett's post‐hoc test for *P < 0.05 and **P < 0.01 compared to Aβ only groups. Scale bar represents 50 μm.

Figure 3

Linagliptin alleviates Aβ‐impaired insulin downstream signaling in SK‐N‐MC neuronal cells. (A) Immunofluorescence images show that the cellular distribution of GLP‐1 receptor is not altered by treatment with Aβ (2.5 μM), linagliptin (50 μM) or in combination for 24 h. (B) Western blotting also reveals that the expression of GLP‐1 receptor is not altered by Aβ (2.5 μM) or linagliptin (50 μM) treatment for 24 h in SK‐N‐MC cells. (C) Immunoblotting reveals that phosphorylation of Tyr‐IRS‐1 and Ser⁴⁷³‐Akt are inhibited when cells are exposed to Aβ (2.5 μM) for 24 h, and this inhibition is effectively restored by linagliptin (50 μM). (D) Western blotting shows that 50 μM of linagliptin‐activated Akt leads to the Ser⁹ phosphorylation of GSK3β, resulting in the inhibition of tau Thr²³¹ phosphorylation by Aβ (2.5 μM) for 24 h. (E) Cell viability is determined by MTT assay, and the linagliptin‐mediated neuroprotective effects are abolished by the co‐treatment of LY294002 (20 μM), a specific inhibitor of PI3‐kinase. All data were performed in three independent experiments, and values are presented as mean ± SEM. Significant differences was determined by using the multiple comparisons of Dunnett's post‐hoc test for *P < 0.05 and **P < 0.01. Scale bar represents 20 μm.

Figure 4

Linagliptin reduces Aβ‐induced intracellular ROS accumulation and improves mitochondria dysfunction. (A) Effects of linagliptin (50 μM) in reducing 2.5 μM of Aβ‐induced intracellular ROS accumulation determined by dichlorofluorescin diacetate (DCFH‐DA) staining under microscope. (B) Effects of linagliptin (50 μM), Aβ (2.5 μM), and LY294002 (20 μM) on Thr¹⁷² phosphorylation of AMPK, and the protein levels of AMPK, Sirt1 and SOD1 by immunoblotting. (C) JC‐1 immunofluorescent staining. Green fluorescence represents Aβ‐induced mitochondrial dysfunction by dissipation of mitochondrial membrane potential. Red fluorescence indicates that co‐treatment with linagliptin (50 μM) preserved an intact mitochondrial membrane potential compared with the group treated with Aβ (2.5 μM) alone. LY294002 (20 μM), a specific inhibitor of PI3‐kinase. Scale bar represents 20 μm.