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. 2017 Mar 15:7:44746.
doi: 10.1038/srep44746.

The endoplasmic reticulum stress/autophagy pathway is involved in cholesterol-induced pancreatic β-cell injury

Fei-Juan Kong  1 , Jia-Hua Wu  1 , Shui-Ya Sun  1 , Jia-Qiang Zhou  1
Affiliations

The endoplasmic reticulum stress/autophagy pathway is involved in cholesterol-induced pancreatic β-cell injury

Fei-Juan Kong et al. Sci Rep. .

Abstract

Lipotoxicity has been implicated in pancreatic β-cell dysfunction in type 2 diabetes, but the exact mechanisms remain unknown. The current study explored the role of the endoplasmic reticulum (ER) stress pathway in cholesterol-induced lipotoxicity. Two different insulinoma cell lines were treated with cholesterol with or without inhibitors. ER stress-associated proteins glucose-regulated protein (GRP) 78, activating transcription factor (ATF) 4 and C/EBP homologous protein (CHOP), as was phosphorylation of eukaryotic initiation factor (EIF) 2α, were all up-regulated by cholesterol. Cholesterol also up-regulated microtubule-associated protein 1 light chain 3 (LC3)-II and stimulated the formation of autophagic vacuoles and LC3-II aggregates. Cholesterol-induced autophagy and cell injuries were suppressed by pretreatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA). Pretreatment with autophagy inhibitors E-64d/pepstatin A increased ER stress-induced cell injuries as indicated by increased cell apoptosis and decreased insulin secretion. These results suggest that cholesterol treatment induces apoptosis and dysfunction of β-cells, and enhances autophagy through activation of the ER stress pathway. More importantly, autophagy induced by cholesterol may protect β-cells against ER stress-associated cell damages.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Cholesterol treatment induces apoptosis in pancreatic β-cells.
Expression of cleaved caspase-3, bcl-2, and bax in INS-1 cells (A–C) and βTC-6 cells (D–F) determined by western blotting. Apoptosis in INS-1 cells was detected using DAPI staining (G,H). Arrows indicate apoptotic cell nuclei. Data are expressed as the mean ± SD values of three to five independent experiments. Statistical significance was assessed using the Student’s t test. *P < 0.05, **P < 0.01 compared to CON. CON = controls; CHO = cholesterol.
Figure 2
Figure 2. Cholesterol treatment activates ER stress in pancreatic β-cells.
Expression of GRP78, p-EIF2α, ATF4 and CHOP in INS-1 cells (A–E) and βTC-6 cells (F–J) determined by western blotting. Data are expressed as the mean ± SD values of three to five independent experiments. Statistical significance was assessed using the Student’s t test. *P < 0.05, **P < 0.01 compared to CON. CON = controls; CHO = cholesterol.
Figure 3
Figure 3. Cholesterol treatment stimulates autophagy in pancreatic β-cells.
Expression of LC3-II in INS-1 and βTC-6 cells determined by western blotting (A–C). Formation of autophagic vacuoles in INS-1 cells was estimated using TEM (D,E). Arrows indicate autophagic vacuoles. Formation of LC3-II point-like aggregates in INS-1 cells evaluated using immunofluorescence (F,G). Data are expressed as the mean ± SD values of three to five independent experiments. Statistical significance was analyzed using the Student’s t test and one-way ANOVA. Statistical significance was determined by *P < 0.05, **P < 0.01 compared to CON, #P < 0.05, ##P < 0.01 compared to CHO. CON = controls; CHO = cholesterol; EP = E64d + pepstatin A.
Figure 4
Figure 4. Inhibition of ER stress attenuates cholesterol-induced β-cell injury and autophagy.
Expression of CHOP, LC3-II and cleaved caspase-3 in INS-1 cells (A–D) and βTC-6 cells (E–H) determined by western blotting. Apoptosis in INS-1 cells was detected using DAPI staining (I,J). Arrows indicate apoptotic cell nuclei. Insulin secretion in βTC-6 cells was evaluated using an ELISA kit and normalized against protein content (K). Data are expressed as the mean ± SD values of three to five independent experiments. Statistical significance was analyzed using a one-way ANOVA followed by Tukey’s multiple comparison testing. Statistical significance was determined by *P < 0.05, **P < 0.01 compared to CON, #P < 0.05, ##P < 0.01 compared to CHO. CON = control; CHO = cholesterol; PBA = 4-phenylbutyrate.
Figure 5
Figure 5. Inhibition of autophagy aggravates ER stress-induced cell injuries.
Expression of cleaved caspase-3, bcl-2, and bax in INS-1 cells (A–C) and βTC-6 cells (D–F) determined by western blotting. Apoptosis in INS-1 cells was detected using DAPI staining (G,H). Arrows indicate apoptotic cell nuclei. Insulin secretion in βTC-6 cells was evaluated using an ELISA kit and normalized against protein content (I). Data are expressed as the mean ± SD values of three to five independent experiments. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s multiple comparison testing. Statistical significance was determined by *P < 0.05, **P < 0.01 compared to CON, #P < 0.05, ##P < 0.01 compared to CHO. CON = controls; CHO = cholesterol; EP = E64d+ pepstatin A.
Figure 6
Figure 6. Model of the pathway of cholesterol-induced ER stress and autophagy in β-cells.
Cholesterol stimulates autophagy and cell injuries in pancreatic β-cell lines, which are associated with the up-regulation or activation of ER stress proteins GRP78, p-EIF2α, ATF4 and CHOP. Inhibition of autophagy further aggravates cell injuries induced by cholesterol.
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