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. 2019 Apr 11:2019:8463125.
doi: 10.1155/2019/8463125. eCollection 2019.

Melatonin Affects Mitochondrial Fission/Fusion Dynamics in the Diabetic Retina

Janet Ya-An Chang  1   2 , Fei Yu  1 , Liheng Shi  1 , Michael L Ko  1 , Gladys Y-P Ko  1   2   3
Affiliations

Melatonin Affects Mitochondrial Fission/Fusion Dynamics in the Diabetic Retina

Janet Ya-An Chang et al. J Diabetes Res. .

Abstract

Mitochondrial fission and fusion are dependent on cellular nutritional states, and maintaining this dynamics is critical for the health of cells. Starvation triggers mitochondrial fusion to maintain bioenergetic efficiency, but during nutrient overloads (as with hyperglycemic conditions), fragmenting mitochondria is a way to store nutrients to avoid waste of energy. In addition to ATP production, mitochondria play an important role in buffering intracellular calcium (Ca2+). We found that in cultured 661W cells, a photoreceptor-derived cell line, hyperglycemic conditions triggered an increase of the expression of dynamin-related protein 1 (DRP1), a protein marker of mitochondrial fission, and a decrease of mitofusin 2 (MFN2), a protein for mitochondrial fusion. Further, these hyperglycemic cells also had decreased mitochondrial Ca2+ but increased cytosolic Ca2+. Treating these hyperglycemic cells with melatonin, a multifaceted antioxidant, averted hyperglycemia-altered mitochondrial fission-and-fusion dynamics and mitochondrial Ca2+ levels. To mimic how people most commonly take melatonin supplements, we gave melatonin to streptozotocin- (STZ-) induced type 1 diabetic mice by daily oral gavage and determined the effects of melatonin on diabetic eyes. We found that melatonin was not able to reverse the STZ-induced systemic hyperglycemic condition, but it prevented STZ-induced damage to the neural retina and retinal microvasculature. The beneficial effects of melatonin in the neural retina in part were through alleviating STZ-caused changes in mitochondrial dynamics and Ca2+ buffering.

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Figures

Figure 1
Figure 1
High glucose induces changes in mitochondrial fission/fusion dynamics in cultured 661W cells. Cultured 661W cells were acutely exposed to high-glucose conditions (HG; 30 mM) for 4, 6, 16, or 24 h (a-d) and collected for Western blotting of DRP1 (b), MFN2 (c), and MCU (d). The control (c) cells were treated with H2O. (e-g) Cultured 661W cells were treated with 0.01% ethanol (vehicle) as the control (CON), melatonin (MEL, 100 μM; dissolved in 0.01% ethanol), HG (30 mM), or a combination of melatonin and HG (HG+MEL) for 24 h. Cells were then collected and processed for Western blotting analysis of DRP1 (f), MFN2 (g), and MCU (h). The experiments were repeated at least four times (n = 4-5). ∗p < 0.05.
Figure 2
Figure 2
Melatonin treatments alleviate HG-induced decreases in mitochondrial Ca2+ buffering. Calcium-imaging of cultured 661W cells after 24 h of treatments with H2O (CON), HG, or HG+melatonin (HG+M). Cells were loaded with Fluo-4 and Rhod-2 for cytosolic and mitochondrial Ca2+ imaging, respectively. Scale bar = 20 μm in (a) and 10 μm in (b). The Rhod-2 (c) and Fluo-4 (d) fluorescent intensities indicating mitochondrial (c) and cytosolic (d) Ca2+ were quantified. The experiments were repeated at least three times. ∗p < 0.05.
Figure 3
Figure 3
Daily treatments with melatonin did not improve STZ-induced diabetic conditions in body weights and systemic hyperglycemia. One week after STZ injections, some STZ-injected mice were given daily melatonin via oral gavage (STZ+MEL). (a) STZ-induced diabetic mice (STZ) with or without melatonin treatments gained weight more slowly starting 1 week after STZ injections compared to the control mice injected with citric buffer (CON). Three months after melatonin treatment, the average body weights of STZ+MEL mice or STZ mice were lower than those of the control mice (∗). There was no statistical difference between the CON and melatonin-treated (MEL) groups. (b) The STZ and STZ+MEL mice had significantly higher systemic blood glucose levels than the CON or MEL (∗). The blood glucose levels of STZ+MEL mice were higher than those of STZ mice (#) after 3 months post-STZ injection. ∗ denotes a statistical significance compared to the control mice (CON); # denotes a statistical significance compared to the STZ mice. ∗, #p < 0.05.
Figure 4
Figure 4
Dark-adapted ERG a-wave amplitudes and implicit times recorded at one, two, and three months after STZ injections. The dark-adapted ERG a-wave amplitudes (a, c, e) and implicit times (b, d, f) in the control (CON), STZ-injected (STZ), melatonin-treated (MEL), and STZ-injected and melatonin-treated (STZ+MEL) mice at 1 month (a, b), 2 months (c, d), and 3 months (e, f) after the STZ injections. ∗ denotes a statistical significance between CON and STZ; # denotes a statistical significance between MEL and STZ+MEL; $ denotes a statistical significance between CON and MEL; ∗p < 0.05.
Figure 5
Figure 5
Dark-adapted ERG b-wave amplitudes and implicit times recorded at one, two, and three months after STZ injections. The dark-adapted ERG b-wave amplitudes (a, c, e) and implicit times (b, d, f) in the control (CON), STZ-injected (STZ), melatonin-treated (MEL), and STZ-injected and melatonin-treated (STZ+MEL) mice at 1 month (a, b), 2 months (c, d), and 3 months (e, f) after the STZ injections. ∗ denotes a statistical significance between CON and STZ; # denotes a statistical significance between MEL and STZ+MEL; $ denotes a statistical significance between CON and MEL; % denotes a statistical significance between STZ and STZ+MEL; ∗p < 0.05.
Figure 6
Figure 6
Melatonin treatments appear to prevent the development of microvascular complications. (a) Fluorescein angiography was used to visualize the intraocular vasculature in mice. AngioTool was used to determine the (b) percentage of vascular area to the retinal area and (c) the average vessel length. “AU” is the arbitrary unit used in the AngioTool software. The vascular area (percentage) and the average vessel length in STZ+MEL mice are significantly less than those in the STZ mice (∗). (d) Venous beading (rectangle) was observed in STZ mice. ∗p < 0.05.
Figure 7
Figure 7
Daily melatonin treatments prevent STZ-induced changes in mitochondrial fusion and Ca2+ uniporter proteins. The immunofluorescent images of DRP1, MFN2, and MCU in the neural retinas from three groups are shown. The analyzed data of relative changes in fluorescent intensities of the whole retina (second row), the photoreceptors (IS only; third row), and the inner retina (from INL to GL; third row) are shown. The fluorescent intensities of the control (CON) were arbitrarily set at 1 for each slide. Each datum point is the average of relative fluorescent intensities (multiple images) from a single mouse. There is no apparent change of DRP1 in the STZ mouse retina (STZ) compared to the control (CON). Melatonin does not have an impact on DRP1 in STZ mouse retinas (STZ+MEL). The STZ mouse retina (STZ) has an apparent decrease in MFN2 compared to both CON and melatonin-treated (STZ+MEL) groups in the photoreceptors. The STZ mouse retina (STZ) also has a significantly decreased MCU expression compared to the melatonin (STZ+MEL) group in the photoreceptors and inner retina. BF = Bright field. DAPI stained nuclei. Scale bar = 50 μm. OS: photoreceptor outer segments; IS: photoreceptor inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GL: ganglion cell layer. ∗p < 0.05.
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