Liraglutide protects palmitate-induced INS-1 cell injury by enhancing autophagy mediated via FoxO1

Abstract

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and a progressive loss in mass and function of pancreatic β-cells. In T2DM, lipotoxicity leads to β-cells dysfunction and decreases its number. Autophagy serves a crucial role in maintaining the normal islet architecture and the function of β-cells. Moreover, glucagon-like peptide-1 (GLP-1) and its analogs have beneficial roles in pancreatic β-cells. However, the protective effects of GLP-1 agents on palmitate (PA)-induced pancreatic β-cells and their underlying mechanisms are not fully elucidated. Forkhead box O1 (FoxO1) can prevent pancreatic β-cells from apoptosis. Whether GLP-1 protects against PA-induced β-cells injury via FoxO1 remains unknown. The present study exposed INS-1 cells to PA to establish a T2DM injury model. Cell viability was evaluated using a Cell Counting Kit-8 assay, and apoptosis was determined via western blotting. Furthermore, autophagy was examined using western blotting, immunofluorescence and transmission electron microscopy. Silencing FoxO1 was used to inhibit the activities of FoxO1. The results suggested that the GLP-1 analog liraglutide enhanced the cell viability, inhibited the protein expression of cleaved caspase-3 and increased the expression levels of microtubule-associated protein 1 light chain3 (LC3) II/I, and FoxO1 in INS-1 cells. The autophagy inhibitor chloroquine inhibited the protective effects of liraglutide on INS-1 cells. Silencing of FoxO1 decreased the expression levels of LC3-II and attenuated the protection of liraglutide on the viability of INS-1 cells. In conclusion, the results indicated that liraglutide ameliorated the PA-induced islet β-cells injury via the upregulation of autophagy-mediated by FoxO1.

Keywords: T2DM; pancreatic β cells; autophagy; liraglutide; FoxO1.

Figure 1.

Effects of palmitate in INS-1 cells injury, autophagy and FoxO1. (A) Cell Counting Kit-8 assay was used to examine the viability of INS-1 cells, which were treated with various concentrations of palmitate (0, 0.1, 0.3 and 0.5 mmol/l). Data were analyzed by unpaired t-test and are presented as the mean ± SD (n=3). *P<0.05 vs. Con group. Western blotting, using GAPDH as a control, was conducted to determine autophagy-related protein (B) LC3 and (C) FoxO1 expression levels. CON, control; FoxO1, Forkhead box O1; LC3, microtubule-associated protein 1 light chain3.

Figure 2.

Effect of autophagy in PA-induced INS-1 cells injury and FoxO1. (A) Cell Counting Kit-8 analysis of the viability of INS-1 cells in each group. Data are presented as the mean ± SD (n=3). ^#P<0.05 vs. CON group; *P<0.05 vs. PA group; ^$P<0.05 vs. LIRA group; ^{^}P<0.05 vs. PA + LIRA group. Data were analyzed by unpaired t-test. (B) Western blotting was used to detect the expression of LC3 in each group. (C) Immunofluorescence analysis of LC3 puncta in each group (magnification, ×100). (D) Micromorphological changes in cellular organelles were examined via transmission electron microscopy. The red arrow points to autophagic vesicles (magnification, ×20,000; n=3). The red circles indicate the presence of autophagic vacuole structures. (E) Western blotting, using GAPDH as a control, was conducted to determine p-FoxO1 and FoxO1 protein expression levels in each group. FoxO1, Forkhead box O1; LC3, microtubule-associated protein 1 light chain3; CON, control; PA, palmitate; CQ, chloroquine; GLP-1, glucagon-like peptide-1; p-, phosphorylated.

Figure 3.

Effect of liraglutide in PA-induced INS-1 cells injury, autophagy and FoxO1. (A) Western blotting, using β-actin as a control, for determining cleaved caspase-3 protein expression. Data were analyzed by paired t-test and are presented as the mean ± SD (n=3). ^#P<0.05 vs. CON group; *P<0.05 vs. PA group. (B) Immunofluorescence analysis of LC3 puncta in control and GLP-1 group. Scale bar, 50 µm. (C). Western blotting was used to detect the expression levels of p-FoxO1 and FoxO1 in each group. FoxO1, Forkhead box O1; LC3, microtubule-associated protein 1 light chain3; CON, control; PA, palmitate; GLP-1, glucagon-like peptide-1; p-, phosphorylated.

Figure 4.

Liraglutide improves the viability and autophagy induced by PA via FoxO1. (A) A Cell Counting Kit-8 assay was used to analyze the viability of INS-1 cells in each group. Data were analyzed by paired t-test and are presented as the mean ± SD (n=3). *P<0.05 vs. GLP-1 + siCON group; ^#P<0.05 vs. PA + siCON group. (B) Western blotting was used to detect the expression levels of FoxO1 and LC3 in each group. FoxO1, Forkhead box O1; LC3, microtubule-associated protein 1 light chain3; CON, control; PA, palmitate; GLP-1, glucagon-like peptide-1; si, small interfering RNA.

Figure 5.

GLP-1 analog liraglutide induces autophagy and improves INS-1 cell survival in palmitate medium by activating FoxO1. Palmitate downregulated FoxO1, caused the dysfunction of autophagy and decreased INS-1 cell survival, thus inducing DM. The autophagy inhibitor chloroquine decreased the viability of INS-1 cells. Moreover, siFoxO1 suppressed autophagy and decreased the protection of liraglutide on the viability of INS-1 cells. Thus, liraglutide protected islet β-cells from T2DM. FoxO1, Forkhead box O1; LC3, microtubule-associated protein 1 light chain3; GLP-1, glucagon-like peptide-1; siRNA, small interfering RNA; T2DM, type 2 diabetes mellitus.