Dapagliflozin Prevents NOX- and SGLT2-Dependent Oxidative Stress in Lens Cells Exposed to Fructose-Induced Diabetes Mellitus

Abstract

Purpose: Cataracts in patients with diabetes mellitus (DM) are a major cause of blindness in developed and developing countries. This study aims to examine whether the generation of reactive oxygen species (ROS) via the increased expression of glucose transporters (GLUTs) and the receptor for advanced glycation end products (RAGE) influences the cataract development in DM.

Methods: Lens epithelial cells (LECs) were isolated during cataract surgery from patients without DM or with DM, but without diabetic retinopathy. In a rat model, fructose (10% fructose, 8 or 12 weeks) with or without dapagliflozin (1.2 mg/day, 2 weeks) treatment did induce DM, as verified by blood pressure and serum parameter measurements. Immunofluorescence stainings and immunoblottings were used to quantify the protein levels. Endogenous O₂˙¯ production in the LECs was determined in vivo with dihydroethidium stainings.

Results: We investigated that GLUT levels in LECs differed significantly, thus leading to the direct enhancement of RAGE-associated superoxide generation in DM patients with cataracts. Superoxide production was significantly higher in LECs from rats with fructose-induced type 2 DM, whereas treatment with the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin prevented this effect in fructose-fed rats. Protein expression levels of the sodium/glucose cotransporter 2 (SGLT2), GLUT1, GLUT5, the nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase subunit p67-phox, NOX2/4 and RAGE were upregulated in fructose-fed animals, whereas dapagliflozin treatment reversed these effects.

Conclusions: In rats with fructose-induced DM, dapagliflozin downregulates RAGE-induced NADPH oxidase expression in LECs via the inactivation of GLUTs and a reduction in ROS generation. These novel findings suggest that the SGLT2 inhibitor dapagliflozin may be a candidate for the pharmacological prevention of cataracts in patients with DM.

Keywords: NADPH oxidase; cataract; dapagliflozin; glucose transporter; type 2 diabetes mellitus.

Figure 1

Glucose transporters (GLUTs) may act through the receptor for advanced glycation end products (RAGE) and induce Ras-related C3 botulinum toxin substrate 1 (RAC1) expression and superoxide generation in the lens tissue of cataract patients with diabetes mellitus (DM). (A) Representative fluorescence images of sodium/glucose cotransporter 2 (SGLT2)- and 3-NT-positive cells (green) and SGLT2-positive cells (red) in lens epithelial tissue from cataract patients with and without DM. Cell nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B,C) Representative fluorescence images of RAGE- and GLUT1-positive cells (green) and RAC1- and GLUT5-positive cells (red) in lens epithelial tissue from cataract patients with and without DM. Cell nuclei are counterstained with DAPI (blue). Scale bar: 20 μm. Quantified values (right) are the means ± SEMs (n = 10 per group, separate experimental groups in each figure). * p < 0.05.

Figure 2

Glucose transporter (GLUT)-induced superoxide production in lenses of rats with fructose-induced type 2 diabetes mellitus (DM). (A) Lenses from the control (a) and fructose (b) groups. Rats exhibited fructose-induced type 2 DM after eight weeks (8 W). (B) Representative images of dihydroethidium-treated lens epithelial cells. Sections from rats with fructose-induced type 2 DM displayed significantly higher dihydroethidium fluorescence compared to the control group, whereas treatment with the sodium-glucose cotransporter 2 (SGLT2) inhibitor (dapagliflozin, 1.2 mg/kg/day) prevented this effect in fructose-fed rats. (C–D) Real-time polymerase chain reaction (RT-PCR) depicting SGLT2, GLUT1, and GLUT5 mRNA expression in the lens epithelium or fibers from animals with fructose-induced type 2 DM with or without dapagliflozin administration. Scale bar: 20 μm. Data are presented as the means ± SEM (n = 6 per group, separate experimental groups). * p < 0.05 vs. control; ^# p < 0.05 vs. Fructose 8 W.

Figure 3

Dapagliflozin reduces glucose transporter (GLUT)-induced expression of the receptor for advanced glycation end products (RAGE), nicotinamide adenine dinucleotide phosphate reduced form oxidase 4 (NOX4) and nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase subunit (p67-phox) levels in the lenses of rats with fructose-induced type 2 diabetes mellitus (DM). (A–B) Representative fluorescence images of GLUT5- and RAGE-expressing cells (green) and p7-phox and NOX4-expressing cells (red) in the lens with or without a systemic administration of fructose or dapagliflozin. Cell nuclei are counterstained with DAPI (blue). (C–E) Quantitative immunoblotting analysis demonstrating that the GLUT1, GLUT5, and p67-phox levels in the lenses of rats with fructose-induced type 2 DM were significantly decreased by dapagliflozin administration. Values are presented as the means ± SEMs (n = 6 per group, separate experimental groups in each figure). Scale bar: 20 μm. * p < 0.05; ^# p < 0.05 vs. Fructose 8 W or 12 W.

Figure 4

Dapagliflozin blocked the sodium-glucose cotransporter 2 (SGLT2)-induced production of the NADPH Oxidase Subunits and the receptor for advanced glycation end products (RAGE) in lens epithelial sections of type 2 diabetes mellitus (DM) rats. (A–B) Representative fluorescence images of 3-nitrotyrosine- (3-NT) and glucose transporter (GLUT)5-expressing cells (green) and SGLT2- and GLUT1-expressing cells (red) in the lens of rats with or without the systemic administration of fructose or dapagliflozin. Cell nuclei are counterstained with DAPI (blue). The presented values are the means ± SEMs (n = 6 per group, separate experimental groups in each figure). (C–D) Quantitative immunoblotting analysis demonstrating that the expression levels of SGLT2, GLUT1, GLUT5, RAGE, NOX2 and p67-phox in the lenses of rats with fructose-induced type 2 DM were decreased by dapagliflozin administration. Scale bar: 20 μm. The presented values are the means ± SEMs (n = 6 per group, separate experimental groups in each figure). * p < 0.05 vs. control; ^# p < 0.05 vs. Fructose 8 W.

Figure 5

Dapagliflozin decreases the fructose-induced NOX2/4-dependent oxidative stress in the lens that is mediated by a sodium-glucose cotransporter 2 (SGLT2)-dependent mechanism in a rat model of type 2 diabetes mellitus (DM). Fructose increases the generation of reactive oxygen species by the SGLT2-induced upregulation of the expression levels for advanced glycation end products (AGE), the receptor for AGE (RAGE), and NADPH oxidase isoforms (NOX2/4) in lens epithelial cells of rats (black line). The SGLT2 inhibitor dapagliflozin acts as an important regulator of fructose by downregulating the SGLT2-induced activity of AGE-RAGE-NOX2/4 (red line).