Acute hypoglycemia induces retinal cell death in mouse

Abstract

Background: Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Glycemic excursions could lead to cardiovascular disease, nephropathy, neuropathy and retinopathy. A vast body of literature exists on hyperglycemia namely in the field of diabetic retinopathy, but very little is known about the deleterious effect of hypoglycemia. Therefore, we decided to study the role of acute hypoglycemia in mouse retina.

Methodology/principal findings: To test effects of hypoglycemia, we performed a 5-hour hyperinsulinemic/hypoglycemic clamp; to exclude an effect of insulin, we made a hyperinsulinemic/euglycemic clamp as control. We then isolated retinas from each group at different time-points after the clamp to analyze cells apoptosis and genes regulation. In parallel, we used 661W photoreceptor cells to confirm in vivo results. We showed herein that hypoglycemia induced retinal cell death in mouse via caspase 3 activation. We then tested the mRNA expression of glutathione transferase omega 1 (Gsto1) and glutathione peroxidase 3 (Gpx3), two genes involved in glutathione (GSH) homeostasis. The expression of both genes was up-regulated by low glucose, leading to a decrease of reduced glutathione (GSH). In vitro experiments confirmed the low-glucose induction of 661W cell death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. Moreover, decrease of GSH content by inhibition with buthionine sulphoximine (BSO) at high glucose induced apoptosis, while complementation with extracellular glutathione ethyl ester (GSHee) at low glucose restored GSH level and reduced apoptosis.

Conclusions/significance: We showed, for the first time, that acute insulin-induced hypoglycemia leads to caspase 3-dependant retinal cell death with a predominant role of GSH content.

Figure 1. Insulin-induced hypoglycemia in C57BL/6 mouse.

A) Graphic representation of plasma glucose levels; B) glucose infusion rates during the hyperinsulinemic/hypoglycemic clamp (black circle) and the control hyperinsulinemic/euglycemic clamp (white circle). C) Mouse characteristics before and during the clamp.

Figure 2. Acute hypoglycemia induced cell death in mouse retina.

A) Flat-mounted retinas were isolated 48 h after the clamp, stained for cell death by TUNEL assay and DAPI counter coloration was performed. White arrows show TUNEL positive cells in hypoglycemic condition. B) Quantification of TUNEL positive cells was performed under a fluorescence microscope on retinal flat-mounts. Results are expressed as mean ± SEM of 3 different retinas for each group, * p<0.006 Hypo vs. Eugly. C) Ten µm-embedded frozen sections of enucleated eyes isolated from control (Ctl), hypoglycemic (Hypo) and euglycemic (Eugly) animals were stained for cell death by colorimetric TUNEL system. Using this procedure, apoptotic nuclei are stained dark brown (black arrow). A representative region of three different isolated retinas is shown.

Figure 3. Low glucose concentration induced 661W cell death.

661W, ARPE19 and Mio-M1 cells were cultured as mentioned in material & methods, then A) cells were incubated at 2 mM glucose for different time periods or for 24 h at different glucose concentrations. ATP content was measured and found decreased in 661W cells (white box) after exposure to low glucose concentrations while no change was observed for ARPE19 (gray box) and Mio-M1 cells (black box). Results are expressed as mean ± SEM of 3 experiments, # p<0.05, * p<0.002 and ** p<0.0001 vs. control (25 mM glucose or time 0). B) 661W cells were cultured as mentioned above, then cell death was analyzed by TUNEL assay, performed 24 or 48 h after low glucose exposure followed by DAPI counter coloration. White arrows indicate TUNEL positive cells and condensed nuclei. TUNEL analysis was representative of 3 distinct experiments. C) 661W cells were stained with AnnexinV-FITC and 7-AAD and analyzed by FACS after exposure to low glucose for 24 h.

Figure 4. Caspase 3 was activated in the retina of hypoglycemic mice and in 661W cells incubated at low glucose condition.

A) Immunohistological staining with cleaved Caspase 3 antibody showed positive cells (white arrow) in the outer nuclear (ONL) and ganglion cell layers (GCL) of the hypoglycemic mice retina. Counterstaining with DAPI was performed to identify the retinal cell layers. Results were representative of three observed retinas for each group. B) Similar immunostaining of cleaved Caspase 3 (white arrows) was performed on 661W cells, cultured during 48 h at 2 mM glucose and additional Caspase 3 activity was assessed in a time course at low glucose (2 mM) experiment, using a 24 h serum starved (SS) cultured condition as positive control. Results are expressed as mean ± SEM of 3 experiments, *p<0.006 and **p<0.0001 vs. Ctrl (time 0). C) Measures of Caspase 3 activity in 661W cells cultured for 48 h at 2 mM glucose in absence or presence of Z-VAD-FMK inhibitor (10 µM). Results are expressed as mean ± SEM of 3 experiments, *p<0.0001. TUNEL assay was performed on similar conditions; white arrows indicated TUNEL positive cells.

Figure 5. Mitochondrial superoxide production was detected after exposure of 661W cells to low glucose.

Detection of low glucose-induced peroxide production was performed on 661W photoreceptor cells with the MitoSOX™ dye. 661W cells were cultured at 25 mM and 2mM glucose for diverse periods of time. Fluorescence intensity was visualized under a confocal microscope with appropriate filters.

Figure 6. GSH content was decreased in low glucose conditions.

A) GSH content was measured in protein lysates obtained from retina of control (white circle), euglycemic (white triangle) and hypoglycemic (black square) mice. Results were expressed as percent of control and as mean ± SEM of 5 to 7 samples. *p<0.025 vs. Eugly and **p<0.01 vs. Ctrl. B) 661W cells were cultured at low (2 mM) and high (25 mM) glucose conditions and GSH content was measured at diverse periods of time. Results were expressed as mean ± SEM of 3–5 experiments, *p<0.03 and **p<0.0002 vs. 25 mM glucose. C) GSH content was measured in 661W cells cultured for 48 h at 25 mM or 2 mM glucose, in absence or in presence of 200 µM buthionine sulphoximine (BSO) or 1mM extracellular glutathione ethyl ester (GSHee). At the same time we measured cell death by TUNEL assay in each condition. White arrows indicated TUNEL positive cells. Results are expressed as mean ± SEM of 3 experiments; *p<0.003 vs. 2 mM glucose without GSHee.

Figure 7. Expression of enzymes implicated in GSH homeostasis was modulated by glucose concentration.

A) We tested the expression of two genes, Gpx3 and Gsto1, in the retina of hypoglycemic and euglycemic animals 48 h after the clamp. We were able to show, by RT-qPCR, the induction of both genes in hypoglycemic conditions. In addition, we incubated retinal explants isolated from C57bl/6 mice for 48 h at low (2 mM) and high (25 mM) glucose conditions and measured Gpx3 and Gsto1 expression in the whole retina. RL8 (ribosomal protein L8) was used as internal control to normalize RNA expression and results are expressed as mean ± SEM of 3 (4 and 12 h) to 8 (48 h) retinas, *p<0.03 and as mean ± SEM of 4 to 6 isolated retina **p<0.02 vs. 25 mM glucose. B) We tested by RT-qPCR the expression of Gpx1, Gpx4, Gsto1 and Nox4 in 661W cells after incubation for diverse periods of time (4, 12 and 48 h) at low (2 mM) and high (25 mM) glucose conditions. RL8 was used as internal control to normalize RNA expression and results are expressed as mean ± SEM of 3 experiments in triplicate.