Abstract
Aim:
Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro.
Methods:
HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells.
Results:
Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 μg/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells.
Conclusion:
The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.
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Acknowledgements
This work was supported by the National Basic Research Program of China (No 2014CB932002), the CAS Strategic Priority Research Program (No XDB14030502), the CAS/SAFEA International Partnership Program for Creative Research Teams, and the National Natural Science Foundation of China (No 81273004, 31470829, 20977093, and 10935009). We thank Professor Daisuke ITO from Keio University (Japan) for providing the seipin plasmids.
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Fan, Hd., Chen, Sp., Sun, Yx. et al. Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation. Acta Pharmacol Sin 36, 497–506 (2015). https://doi.org/10.1038/aps.2014.164
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DOI: https://doi.org/10.1038/aps.2014.164
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