Neuroprotective Role of GLP-1 Analog for Retinal Ganglion Cells via PINK1/Parkin-Mediated Mitophagy in Diabetic Retinopathy

Abstract

GLP-1 analogs have been widely used to treat patients with type 2 diabetes in recent years and studies have found that GLP-1 analogs have multiple organ benefits. However, the role of GLP-1 analogs in diabetic retinopathy (DR), a common complication of diabetes mellitus (DM), remains controversial. Retinal ganglion cells (RGCs) are the only afferent neurons responsible for transmitting visual information to the visual center and are vulnerable in the early stage of DR. Protection of RGC is vital for visual function. The incretin glucagon-like peptide-1 (GLP-1), which is secreted by L-cells after food ingestion, could lower blood glucose level through stimulating the release of insulin. In the present study, we evaluated the effects of GLP-1 analog on RGCs both in vitro and in vivo. We established diabetic rat models in vivo and applied an RGC-5 cell line in vitro. The results showed that in high glucose conditions, GLP-1 analog alleviated the damage of RGCs. In addition, GLP-1 analog prevented mitophagy through the PINK1/Parkin pathway. Here we demonstrated the neuroprotective effect of GLP-1 analog, which may be beneficial for retinal function, and we further elucidated a novel mechanism in GLP-1 analog-regulated protection of the retina. These findings may expand the multi-organ benefits of GLP-1 analogs and provide new insights for the prevention of DR.

Keywords: GLP-1; GLP-1 analog; diabetic retinopathy; mitophagy; retinal ganglion cell.

FIGURE 1

Liraglutide mitigated high glucose-induced RGCs injuries in vitro. As shown in the figure, RGCs were treated with different concentrations of glucose (25, 40, 65, 80, 95, 110 mmol/L) and different doses of liraglutide (0, 1, 10, 50, 100, 500, and 1000 nmol/L) for 24 h (A–C) Cell viability was detected with CCK-8 assay kits. Values are presented as mean ± SEM. n = 3 independent experiments. *p < 0.05, ***p < 0.001.

FIGURE 2

Liraglutide alleviated high glucose-induced mitochondrial damage in vitro. RGCs were respectively treated with normal glucose (25 mmol/L), high glucose (65 mmol/L), high glucose (65 mmol/L) and liraglutide (10 nmol/L) for 24 h. (A) Morphology changes of RGCs were observed by TEM. Scale bar: 2.0 μm. ►, autophagosomes; mitochondria; N, nucleus. (B) The generation of reactive oxygen species (ROS) in RGCs was detected by fluorescent probes DCFH-DA. Scale bar: 400 μm. (C) Mitochondrial membrane potential (MMP) was detected by fluorescent probes JC-1. Scale bar: 400 μm.

FIGURE 3

Mitophagy in RGCs was alleviated by liraglutide in vitro. As shown in the figure, RGCs were respectively treated with normal glucose (25 mmol/L), high glucose (65 mmol/L), high glucose (65 mmol/L) and liraglutide (10 nmol/L) for 24 h. (A) Relative LC3A/B and p62 expression was determined by Western blot. (B,C) Quantification of LC3A/B and p62 expression. (D) Relative LC3A/B and p62 expression was determined by immunocytochemistry. Scale bar: 100 μm. (E) The fluorescence expression of LC3A/B. Scale bar: 200 μm. Values are presented as mean ± SEM. n = 3 independent experiments. *p < 0.05, **p＜0.01, ***p < 0.001. A.U., arbitrary units.

FIGURE 4

Liraglutide reduced high glucose-induced RGCs damages through mitophagy in vitro. As shown in the figure, RGCs were respectively treated with normal glucose (25 mmol/L), high glucose (65 mmol/L), high glucose (65 mmol/L), and liraglutide (10 nmol/L) for 24 h. Additionally, RGCs were co-treated with high glucose (65 mmol/L), liraglutide (10 nmol/L), and rapamycin for 24 h. (A) Relative LC3A/B and p62 expression was determined by Western blot. (B,C) Quantification of LC3A/B and p62. (D) Cell viability was detected with CCK-8 assay kits. Values are presented as mean ± SEM. n = 3 independent experiments. *p < 0.05, **p＜0.01, ***p < 0.001.

FIGURE 5

Liraglutide weakened RGCs mitophagy via PINK1/Parkin pathway in vitro. RGCs were respectively treated with normal glucose (25 mmol/L), high glucose (65 mmol/L), high glucose (65 mmol/L), and liraglutide (10 nmol/L) for 24 h. (A) Relative PINK1 and Parkin expression was determined by Western blot. (B,C) Quantification of PINK1 and Parkin expression. (D) Relative LC3A/B and p62 expression was determined by immunocytochemistry. Scale bar: 100 μm. Values are presented as mean ± SEM. n = 3 independent experiments. *p < 0.05, **p＜0.01, ***p < 0.001.

FIGURE 6

Liraglutide attenuated RGCs and mitochondrialinjuries in diabetic rats. (A) The beneficial effect of liraglutide on retinal morphological analysis was detected by H&E staining. Scale bar: 40 μm. (B) Morphology changes of RGCs were observed by TEM. Scale bar: 5.0 μm. ►, autophagosomes; , mitochondria; N, nucleus; NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

FIGURE 7

Mitophagy in RGCs was suppressed by liraglutide in diabetic rats. (A,B) Relative LC3A/B and p62 expression was determined by immunohistochemistry (IHC). Scale bar: 40 μm. (C,D) Mean optical density analysis of LC3A/B and p62 expression in RGCs in different groups. Values are presented as mean ± SEM. n = 5. *p < 0.05, **p＜0.01, ***p < 0.001. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

FIGURE 8

Liraglutide regulated PINK1/Parkin pathway of RGCs in diabetic rats. (A,B) Relative PINK1 and Parkin expression was determined by IHC. Scale bar: 40 μm. (C,D) Mean optical density analysis of PINK1 and Parkin expression in RGCs in different groups. Values are presented as mean ± SEM. n = 5. *p < 0.05, ***p < 0.001. NFL: nerve fiber layer. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

FIGURE 9

Protective role of GLP-1 analog for retinal ganglion cells via PINK1/Parkin-mediated mitophagy in diabetic retinopathy.