Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 May 9;19(5):1416.
doi: 10.3390/ijms19051416.

Glucosamine-Induced Autophagy through AMPK⁻mTOR Pathway Attenuates Lipofuscin-Like Autofluorescence in Human Retinal Pigment Epithelial Cells In Vitro

Ching-Long Chen  1 , Yi-Hao Chen  2 , Chang-Min Liang  3 , Ming-Cheng Tai  4 , Da-Wen Lu  5 , Jiann-Torng Chen  6
Affiliations

Glucosamine-Induced Autophagy through AMPK⁻mTOR Pathway Attenuates Lipofuscin-Like Autofluorescence in Human Retinal Pigment Epithelial Cells In Vitro

Ching-Long Chen et al. Int J Mol Sci. .

Abstract

Age-related macular degeneration (AMD) is a vision-threatening age-associated disease. The retinal pigment epithelial (RPE) cells phagocytose and digest photoreceptor outer segment (POS). Incomplete digestion of POS leads to lipofuscin accumulation, which contributes to the pathology of the AMD. Autophagy could help reduce the amount of lipofuscin accumulation. In the present study, we evaluated the effects of glucosamine (GlcN), a natural supplement, on the induction of autophagy and POS-derived lipofuscin-like autofluorescence (LLAF) in ARPE-19 cells in vitro, and investigated the potential molecular pathway involved. Our results revealed that GlcN had no effect on phagocytosis of POS at the lower doses. GlcN treatment induced autophagy in cells. GlcN decreased the LLAF in native POS-treated cells, whereas malondialdehyde or 4-hydroxynonenal-modified POS attenuated this effect. 3-Methyladenine inhibited GlcN-induced autophagy and attenuated the effect of GlcN on the decrease of the native POS-derived LLAF. Furthermore, GlcN induced the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR), whereas Compound C inhibited these effects of GlcN. Altogether, these results suggest that GlcN decreased the native POS-derived LLAF through induction of autophagy, at least in part, by the AMPK⁻mTOR pathway. This mechanism has potential for the preventive treatment of lipofuscin-related retinal degeneration such as AMD.

Keywords: autophagy; glucosamine; lipofuscin; retinal pigment epithelial cells.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of ZO-1, RPE65, and MerTK protein after one- and seven-day cultures in ARPE-19 cells. (A) Western blot analysis detecting the protein expression of ZO-1, RPE65, and MerTK in post-confluent cultures of ARPE-19 cells. The cells were cultured for either one day or seven days. Whole-cell lysates were prepared and analyzed with immunoblotting using anti-ZO-1, anti-RPE65, anti-MerTK, and anti-GAPDH antibodies. (B) Quantification of protein expression levels of ZO-1, RPE65, and MerTK. The optical density of the Western blot bands obtained for ZO-1, RPE65, MerTK, and GAPDH were analyzed. The results are represented as the mean ± SEM. The differences in the protein level of ZO-1, RPE65, and MerTK between groups were compared using the paired t test. *** p < 0.001. (C) After one and seven days of culture, the characteristics of RPE cells, including tight junction proteins (ZO-1) and differentiation markers (RPE65) were identified by immunofluorescence staining. Magnification, ×400. Scale bar: 20 μm. Effects of glucosamine (GlcN) on phagocytosis of POS in ARPE-19 cells. (D) After seven days of cultures, ARPE-19 cells were pre-treated with or without GlcN (2.5, 5, 10, and 20 mM) for 24 h, followed by co-treatment with fluorescein isothiocyanate-labeled POS (FITC–POS) and the indicated concentration of GlcN (2.5, 5, 10, and 20 mM) for 3 h. The fluorescence intensity was measured using a microplate reader and normalized to the control group. The data are represented as mean ± SEM. ns, not significant; ** p < 0.01 versus POS group. (E,F) After seven days of culture, cells were pre-treated with or without GlcN (2.5, 5, 10, and 20 mM) for 24 h, and then co-treated with FITC–POS and the indicated concentration of GlcN (2.5, 5, and 10 mM) for: 3 h (E); and 24 h (F). The mean fluorescence intensity was measured by flow cytometry and normalized to control cells. The data are represented as mean ± SEM; ns, not significant versus POS group.
Figure 2
Figure 2
Glucosamine (GlcN) increases autophagosome and autophagic markers in ARPE-19 cells. (A) The number of monodansylcadaverine (MDC)-labeled vacuoles increased by either GlcN or rapamycin (Rapa) treatment in ARPE-19 cells. Cells were treated with or without 2.5 mM GlcN, 5 mM GlcN, 10 mM GlcN, or 300 nM rapamycin (Rapa) for 18 h and then stained with MDC. Rapa treatment was used as a positive control. MDC-labeled vacuoles were examined by fluorescence microscopy. Magnification, ×400; Scale bar: 20 μm. (B) The number of cytoplasmic LC3 puncta increased by either GlcN or Rapa treatment in ARPE-19 cells. Cells were treated with or without 2.5 mM GlcN, 5 mM GlcN, 10 mM GlcN, or 300 nM rapamycin (Rapa) for 18 h and then stained with anti-LC3 antibody. The cytoplasmic LC3 puncta were examined by fluorescence microscopy. Nuclei were counterstained with DAPI. Magnification, ×400; Scale bar: 20 μm. (C) Effect of GlcN on the expression of LC3-II and p62 protein in ARPE-19 cells. Cells were treated with the indicated concentrations of GlcN (0, 2.5, 5, and 10 mM) for 18 h. Whole-cell lysates were prepared and analyzed with immunoblotting using anti-LC3, anti-p62, and anti-GAPDH antibodies. (D) Optical density of the Western blot bands for LC3-I, LC3-II, p62, and GAPDH was analyzed. The results are represented as the mean ± SEM. The differences in the LC3-II/LC3-I ratios and p62/GAPDH in ARPE-19 cells between the groups were compared using ANOVA. Tukey’s test was used for the post hoc analysis; *** p < 0.001 versus the control group; # p < 0.05; ### p < 0.001. (E) The cells were treated with 5 mM GlcN for the indicated time (0, 2, 4, 6, 18, and 24 h). Whole-cell lysates were prepared and analyzed by immunoblotting using anti-LC3, anti-p62, and anti-GAPDH antibodies. (F) Optical density of the Western blot bands for LC3-I, LC3-II, p62, and GAPDH was analyzed. The results are represented as the mean ± SEM. The differences in the LC3-II/LC3-I ratios and p62/GAPDH in ARPE-19 cells between the groups were compared using ANOVA. Tukey’s test was used for the post hoc analysis; ns, not significant; ** p < 0.01 versus the control group; *** p < 0.001 versus the control group; ### p < 0.001. (G) The cells were treated with 5 mM GlcN for the indicated time (0, 2, 4, 6, 18, and 24 h). The levels of p62 mRNA were quantified by using qPCR. The results are represented as the mean ± SEM. The relative p62 mRNA expression in ARPE-19 cells between the groups were compared using ANOVA. Tukey’s test was used for the post hoc analysis; ns, not significant; * p < 0.05 versus the control group; *** p < 0.001 versus the control group; ### p < 0.001.
Figure 3
Figure 3
GlcN increases autophagy flux in ARPE-19 cells. (A) The cells were pre-treated with or without Bafilomycin A1 (Baf A1; 100 nM) for 1 h followed by co-treatment with or without GlcN (5 mM) for another 18 h. Whole-cell lysates were prepared and analyzed with immunoblotting using anti-LC3, anti-p62, and anti-GAPDH antibodies. (B) The optical density of Western blot bands for LC3-I, LC3-II, p62, and GAPDH was analyzed. The results are represented as the mean ± SEM. The differences in the LC3-II/LC3-I ratios and p62/GAPDH in ARPE-19 cells between the groups were compared using ANOVA. Tukey’s test was used for the post hoc analysis; *** p < 0.001 versus the control group; # p < 0.05; ### p < 0.001.
Figure 4
Figure 4
Effects of glucosamine (GlcN) on photoreceptor outer segment (POS)-derived lipofuscin-like autofluorescence (LLAF) in ARPE-19 cells. (A) Effect of GlcN on native POS-derived LLAF in ARPE-19 cells. Cells were examined by confocal microscopy following seven days of treatment with native POS, native POS with GlcN (5 or 10 mM), or native POS with rapamycin (Rapa; 300 nM), and were compared with untreated cells. Magnification, ×200. Scale bar: 50 μM. (B) Effect of GlcN on either native or modified POS-derived LLAF in ARPE-19 cells. Cells were treated with GlcN (5 or 10 mM), native POS, native POS + GlcN (5 or 10 mM), MDA-modified POS, HNE-modified POS, MDA-modified POS + GlcN (10 mM), or HNE-modified POS + GlcN (10 mM), and were compared with untreated cells. The mean fluorescence intensity (MFI) was quantified by flow cytometry following seven days of incubation. The results are represented as mean ± SEM. The differences of the MFI in ARPE-19 cells were compared between groups using ANOVA, and Tukey’s test was used for post hoc analysis; ns, not significant; ** p < 0.01 versus the control group; *** p < 0.001 versus the control group; # p < 0.05 versus the native POS group; ## p < 0.01 versus the native POS group.
Figure 5
Figure 5
3-Methyladenine (3-MA) inhibited glucosamine (GlcN)-induced autophagy in ARPE-19 cells. (A) The cells were pretreated with or without 3-MA (10 mM) for 1 h followed by co-treatment with or without GlcN (5 mM) for another 18 h. Whole-cell lysates were prepared and analyzed with immunoblotting using anti-LC3 and anti-GAPDH antibodies. (B) Optical density of the Western blot bands for LC3-I, LC3-II, and GAPDH was analyzed. The results are represented as the mean ± SEM. The differences in the LC3-II/LC3-I ratios between groups were compared using ANOVA, and Tukey’s test was used for post hoc analysis; ns, not significant; ** p < 0.01 versus the control group; *** p < 0.001 versus the control group; ### p < 0.001.
Figure 6
Figure 6
3-Methyladenine (3-MA) attenuated the effect of glucosamine (GlcN) on native photoreceptor outer segment (POS)-derived lipofuscin-like autofluorescence (LLAF) in ARPE-19 cells. Cells were treated with GlcN (10 mM), 3-MA (10 mM), native POS, native POS + 3-MA, native POS + GlcN, or GlcN + native POS + 3-MA, and were compared with untreated cells. The mean fluorescence intensity (MFI) was quantified by flow cytometry following seven days of incubation. The results are represented as the mean ± SEM. The differences of the MFI between groups were compared using ANOVA, and Tukey’s test was used for the post hoc analysis; ns, not significant; * p < 0.05 versus the control group; *** p < 0.001 versus the control group; # p < 0.05; ### p < 0.001.
Figure 7
Figure 7
Glucosamine (GlcN)-induced autophagy via the AMPK–mTOR pathway. (A) GlcN decreased the level of p-mTOR protein in a dose-dependent manner in ARPE-19 cells. Cells were treated with the indicated concentrations of GlcN (0, 2.5, or 5 mM) for 18 h. Whole-cell lysates were prepared and analyzed by immunoblotting using anti-phospho-mTOR, anti-mTOR, and anti-GAPDH antibody. (B) The optical density of Western blot bands for p-mTOR, mTOR, and GAPDH was analyzed. The results are represented as the mean ± SEM. The differences in the p-mTOR/mTOR ratio between groups were compared using ANOVA, and Tukey’s test was used for post hoc analysis; *** p < 0.001 versus the control group; ### p < 0.001. (C) GlcN increased the level of p-AMPK protein in a dose-dependent manner. Cells were treated with the indicated concentrations of GlcN (0, 2.5, or 5 mM) for 18 h. Whole-cell lysates were prepared and analyzed with immunoblotting using anti-phospho-AMPK, anti-AMPK, and anti-GAPDH antibodies. (D) The optical density of Western blot bands for p-AMPK, AMPK, and GAPDH was analyzed. The results are represented as the mean ± SEM. The differences in the p-AMPK/AMPK ratio between groups were compared using ANOVA, and Tukey’s test was used for post hoc analysis; *** p < 0.001 versus the control group; ### p < 0.001. (E) GlcN-induced autophagy through the AMPK–mTOR pathway. Cells were pre-treated with or without compound C (Comp C; 5 µM) for 1 h followed by co-treatment with or without GlcN (5 mM) for another 18 h. Whole-cell lysates were prepared and analyzed with immunoblotting using anti-phospho-AMPK, anti-AMPK, anti-phospho-mTOR, anti-mTOR, anti-LC3, and anti-GAPDH antibodies. (F) The optical density of the Western blot bands for p-AMPK, AMPK, p-mTOR, mTOR, LC3-I, LC3-II, and GAPDH was analyzed. The results are represented as the mean ± SEM. The differences in the p-AMPK/AMPK, p-mTOR/mTOR, and LC3-II/LC3-I ratios between groups were compared using ANOVA, and Tukey’s test was used for post hoc analysis; *** p < 0.001.
Figure 8
Figure 8
Schematic representation of the Glucosamine (GlcN)-induced autophagy on the native photoreceptor outer segment (POS)-derived lipofuscin-like autofluorescence (LLAF) and the potential pathway associated with GlcN-induced autophagy in ARPE-19 cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jager R.D., Mieler W.F., Miller J.W. Age-related macular degeneration. N. Engl. J. Med. 2008;358:2606–2617. doi: 10.1056/NEJMra0801537. - DOI - PubMed
    1. Owen C.G., Jarrar Z., Wormald R., Cook D.G., Fletcher A.E., Rudnicka A.R. The estimated prevalence and incidence of late stage age related macular degeneration in the UK. Br. J. Ophthalmol. 2012;96:752–756. doi: 10.1136/bjophthalmol-2011-301109. - DOI - PMC - PubMed
    1. Ferris F.L., 3rd, Fine S.L., Hyman L. Age-related macular degeneration and blindness due to neovascular maculopathy. Arch. Ophthalmol. 1984;102:1640–1642. doi: 10.1001/archopht.1984.01040031330019. - DOI - PubMed
    1. Bird A.C., Bressler N.M., Bressler S.B., Chisholm I.H., Coscas G., Davis M.D., de Jong P.T., Klaver C.C., Klein B.E., Klein R., et al. An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group. Surv. Ophthalmol. 1995;39:367–374. doi: 10.1016/S0039-6257(05)80092-X. - DOI - PubMed
    1. Velez-Montoya R., Oliver S.C., Olson J.L., Fine S.L., Quiroz-Mercado H., Mandava N. Current knowledge and trends in age-related macular degeneration: Genetics, epidemiology, and prevention. Retina. 2014;34:423–441. doi: 10.1097/IAE.0000000000000036. - DOI - PubMed

MeSH terms

LinkOut - more resources