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Analysis of SCAP N-glycosylation and Trafficking in Human Cells
Published on: November 8, 2016
Sirt6 opposes glycochenodeoxycholate-induced apoptosis of biliary epithelial cells through the AMPK/PGC-1α pathway
Jiye Li1,2,3,4, Dongsheng Yu2,3,5, Sanyang Chen1,2,3
11Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052 Henan China.
Background:
Induction of biliary epithelial cell apoptosis by toxic bile acids is involved in the development of cholestatic disease, but the underlying molecular mechanism is not clear. The purpose of this study was to investigate the molecular mechanisms involved in Sirt6 protection against the apoptosis of human intrahepatic biliary epithelial cells (HiBEC) induced by the bile acid glycochenodeoxycholate (GCDC).
Results:
Sirt6 was either overexpressed or knocked down in HiBEC, with or without GCDC pretreatment. The CCK-8 assay was used to assess cell viability and, Hoechst 33258 staining was used to determine apoptotic rate. Mitochondrial DNA (mtDNA) copy number, malondialdehyde (MDA) and reactive oxygen species (ROS) production were detected to evaluate the severity of the mitochondrial dysfunction and oxidative stress. The mRNA and protein levels of PGC-1α, Nrf1, and Nrf2 were analyzed using RT-qPCR and western blot assay. The results showed that Sirt6 opposed GCDC-induced apoptosis in HiBEC via up-regulating PGC-1α expression and stabilizing mtDNA. We used agonists and inhibitors of AMPK to demonstrate that Sirt6 increased PGC-1α expression through the AMPK pathway whereas GCDC had the opposite effect. Finally, western blot, luciferase assay, and co-immunoprecipitation were used to describe a direct interaction and acetylation modification of PGC-1α by Sirt6.
Conclusion:
Our data illuminated that Sirt6 ameliorated GCDC-induced HiBEC apoptosis by upregulating PGC-1α expression through the AMPK pathway and its deacetylation effect.
Insights
Sirtuin 6 (Sirt6) protects human intrahepatic biliary epithelial cells (HiBEC) from toxic bile acid-induced apoptosis by upregulating PGC-1α expression via the AMPK pathway and deacetylation.
Area of Science:
- Cell Biology
- Molecular Biology
- Hepatology
Background:
- Toxic bile acids induce biliary epithelial cell apoptosis, contributing to cholestatic liver diseases.
- The precise molecular mechanisms underlying this process remain unclear.
Purpose of the Study:
- To investigate the protective mechanisms of Sirtuin 6 (Sirt6) against glycochenodeoxycholate (GCDC)-induced apoptosis in human intrahepatic biliary epithelial cells (HiBEC).
Main Methods:
- Cell viability assays (CCK-8), apoptosis assessment (Hoechst staining), mitochondrial dysfunction and oxidative stress evaluation (mtDNA copy number, MDA, ROS).
- Analysis of PGC-1α, Nrf1, and Nrf2 expression (RT-qPCR, Western blot).
- Investigation of the AMPK pathway and Sirt6-PGC-1α interaction (agonist/inhibitor studies, Western blot, luciferase assay, co-immunoprecipitation).
Main Results:
- Sirt6 overexpression inhibited GCDC-induced HiBEC apoptosis, while knockdown exacerbated it.
- Sirt6 stabilized mitochondrial DNA (mtDNA) and reduced oxidative stress.
- Sirt6 upregulated PGC-1α expression via the AMPK pathway and directly interacted with PGC-1α, deacetylating it.
Conclusions:
- Sirt6 confers protection against GCDC-induced apoptosis in HiBEC.
- This protection is mediated by the upregulation of PGC-1α expression through the AMPK pathway and Sirt6's deacetylation of PGC-1α.