Glucosamine-Induced Autophagy through AMPK–mTOR Pathway Attenuates Lipofuscin-Like Autofluorescence in Human Retinal Pigment Epithelial Cells In Vitro
Abstract
:1. Introduction
2. Results
2.1. Expression of ZO-1, RPE65, and MerTK Protein in ARPE-19 Cells after One Day and Seven Days of Culture
2.2. Effect of GlcN on Phagocytosis of POS in ARPE-19 Cells
2.3. GlcN Induces Autophagy in ARPE-19 Cells
2.3.1. Effect of GlcN on the Autophagosomes and Autophagic Markers in ARPE-19 Cells
2.3.2. Effect of GlcN on Autophagic Flux in ARPE-19 Cells
2.4. Effect of GlcN on POS-Derived LLAF in ARPE-19 Cells
2.5. Relationship between GlcN-Induced Autophagy and Attenuating Effects of GlcN on Native POS-Derived LLAF in ARPE-19 Cells
2.5.1. Effects of 3-MA on GlcN-Induced Autophagy
2.5.2. Effects of 3-MA on the Attenuating Effects of GlcN in Native POS-Derived LLAF
2.6. GlcN-Inducted Autophagy via the AMPK–mTOR Pathway
3. Discussion
4. Materials and Methods
4.1. RPE Cell Culture
4.2. RPE Cell Treatment
4.3. Western Blot Analysis
4.4. Immunocytochemistry
4.5. Monodansylcadaverine Staining
4.6. RNA Isolation and Quantitative Polymerase Chain Reaction (qPCR)
4.7. POS Isolation
4.8. Malondialdehyde and 4-Hydroxynonenal Modification of POS
4.9. Phagocytosis Assay
4.10. Flow Cytometry-Based Phagocytosis Assay
4.11. Induction of POS-Derived LLAF in Vitro
4.12. Observation of LLAF in Vitro by Confocal Microscopy
4.13. Quantification of LLAF by Flow Cytometry
4.14. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AMD | Age-related macular degeneration |
RPE cells | Retinal pigment epithelial cells |
POS | Photoreceptor outer segment |
GlcN | Glucosamine |
LLAF | Lipofuscin-like autofluorescence |
GA | Geographic atrophy |
ROS | Reactive oxygen species |
ZO-1 | Zonula occludens-1 |
RPE65 | Retinal pigment epithelium-specific protein 65 kDa |
MerTK | Mer tyrosine kinase |
GAPDH | Glyceraldehyde 3-phosphate dehydrogenase |
MDC | Monodansylcadaverine |
LC3 | Microtubule-associated protein 1 light chain 3 |
p62 | sequestosome 1 |
Rapa | Rapamycin |
Baf A1 | Bafilomycin A1 |
MDA | Malondialdehyde |
HNE | 4-hydroxynonenal |
3-MA | 3-Methyladenine |
AMPK | AMP-activated protein kinase |
mTOR | mammalian target of rapamycin |
Comp C | Compound C |
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Chen, C.-L.; Chen, Y.-H.; Liang, C.-M.; Tai, M.-C.; Lu, D.-W.; Chen, J.-T. Glucosamine-Induced Autophagy through AMPK–mTOR Pathway Attenuates Lipofuscin-Like Autofluorescence in Human Retinal Pigment Epithelial Cells In Vitro. Int. J. Mol. Sci. 2018, 19, 1416. https://doi.org/10.3390/ijms19051416
Chen C-L, Chen Y-H, Liang C-M, Tai M-C, Lu D-W, Chen J-T. Glucosamine-Induced Autophagy through AMPK–mTOR Pathway Attenuates Lipofuscin-Like Autofluorescence in Human Retinal Pigment Epithelial Cells In Vitro. International Journal of Molecular Sciences. 2018; 19(5):1416. https://doi.org/10.3390/ijms19051416
Chicago/Turabian StyleChen, Ching-Long, Yi-Hao Chen, Chang-Min Liang, Ming-Cheng Tai, Da-Wen Lu, and Jiann-Torng Chen. 2018. "Glucosamine-Induced Autophagy through AMPK–mTOR Pathway Attenuates Lipofuscin-Like Autofluorescence in Human Retinal Pigment Epithelial Cells In Vitro" International Journal of Molecular Sciences 19, no. 5: 1416. https://doi.org/10.3390/ijms19051416