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

Ying-Ying Chen, Tsung-Tien Wu, Chiu-Yi Ho, Tung-Chen Yeh, Gwo-Ching Sun, Ya-Hsin Kung, Tzyy-Yue Wong, Ching-Jiunn Tseng, Pei-Wen Cheng, Ying-Ying Chen, Tsung-Tien Wu, Chiu-Yi Ho, Tung-Chen Yeh, Gwo-Ching Sun, Ya-Hsin Kung, Tzyy-Yue Wong, Ching-Jiunn Tseng, Pei-Wen Cheng

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.

Conflict of interest statement

The authors of this study declared no conflicts of interest.

Figures

Figure 1
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
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
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
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
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).

References

    1. Tsilas C.S., de Souza R.J., Mejia S.B., Mirrahimi A., Cozma A.I., Jayalath V.H., Ha V., Tawfik R., Di Buono M., Leiter L.A. Relation of total sugars, fructose and sucrose with incident type 2 diabetes: A systematic review and meta-analysis of prospective cohort studies. CMAJ. 2017;189:E711–E720. doi: 10.1503/cmaj.160706.
    1. Maarman G.J., Mendham A.E., Lamont K., George C. Review of a causal role of fructose-containing sugars in myocardial susceptibility to ischemia/reperfusion injury. Nutr. Res. 2017;42:11–19. doi: 10.1016/j.nutres.2017.03.003.
    1. Pollreisz A., Schmidt-Erfurth U. Diabetic cataract-pathogenesis, epidemiology and treatment. J. Ophthalmol. 2010;2010:608751. doi: 10.1155/2010/608751.
    1. Zhang S., Chai F.Y., Yan H., Guo Y., Harding J.J. Effects of N-acetylcysteine and glutathione ethyl ester drops on streptozotocin-induced diabetic cataract in rats. Mol. Vis. 2008;14:862–870.
    1. Kumar P.A., Reddy P.Y., Srinivas P.N., Reddy G.B. Delay of diabetic cataract in rats by the antiglycating potential of cumin through modulation of alpha-crystallin chaperone activity. J. Nutr. Biochem. 2009;20:553–562. doi: 10.1016/j.jnutbio.2008.05.015.
    1. Cheng P.W., Chen Y.Y., Cheng W.H., Lu P.J., Chen H.H., Chen B.R., Yeh T.C., Sun G.C., Hsiao M., Tseng C.J. Wnt Signaling Regulates Blood Pressure by Downregulating a GSK-3beta-Mediated Pathway to Enhance Insulin Signaling in the Central Nervous System. Diabetes. 2015;64:3413–3424. doi: 10.2337/db14-1439.
    1. Francini F., Castro M.C., Schinella G., Garcia M.E., Maiztegui B., Raschia M.A., Gagliardino J.J., Massa M.L. Changes induced by a fructose-rich diet on hepatic metabolism and the antioxidant system. Life Sci. 2010;86:965–971. doi: 10.1016/j.lfs.2010.05.005.
    1. Yeh T.C., Liu C.P., Cheng W.H., Chen B.R., Lu P.J., Cheng P.W., Ho W.Y., Sun G.C., Liou J.C., Tseng C.J. Caffeine intake improves fructose-induced hypertension and insulin resistance by enhancing central insulin signaling. Hypertension. 2014;63:535–541. doi: 10.1161/HYPERTENSIONAHA.113.02272.
    1. Bendall J.K., Rinze R., Adlam D., Tatham A.L., de Bono J., Wilson N., Volpi E., Channon K.M. Endothelial Nox2 overexpression potentiates vascular oxidative stress and hemodynamic response to angiotensin II: Studies in endothelial-targeted Nox2 transgenic mice. Circ. Res. 2007;100:1016–1025. doi: 10.1161/01.RES.0000263381.83835.7b.
    1. Guglielmotto M., Aragno M., Tamagno E., Vercellinatto I., Visentin S., Medana C., Catalano M.G., Smith M.A., Perry G., Danni O., et al. AGEs/RAGE complex upregulates BACE1 via NF-kappaB pathway activation. Neurobiol. Aging. 2012;33:196-e13. doi: 10.1016/j.neurobiolaging.2010.05.026.
    1. Miller A., Adeli K. Dietary fructose and the metabolic syndrome. Curr. Opin. Gastroenterol. 2008;24:204–209. doi: 10.1097/MOG.0b013e3282f3f4c4.
    1. Wautier M.P., Chappey O., Corda S., Stern D.M., Schmidt A.M., Wautier J.L. Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE. Am. J. Physiol. Endocrinol. Metab. 2001;280:E685–E694. doi: 10.1152/ajpendo.2001.280.5.E685.
    1. Wilder-Smith C.H., Li X., Ho S.S., Leong S.M., Wong R.K., Koay E.S., Ferraris R.P. Fructose transporters GLUT5 and GLUT2 expression in adult patients with fructose intolerance. United Eur. Gastroenterol. J. 2014;2:14–21. doi: 10.1177/2050640613505279.
    1. Mantych G.J., Hageman G.S., Devaskar S.U. Characterization of glucose transporter isoforms in the adult and developing human eye. Endocrinology. 1993;133:600–607. doi: 10.1210/endo.133.2.8344201.
    1. Nauck M.A. Update on developments with SGLT2 inhibitors in the management of type 2 diabetes. Drug Des. Dev. Ther. 2014;8:1335–1380. doi: 10.2147/DDDT.S50773.
    1. Liu X., Luo D., Zheng M., Hao Y., Hou L., Zhang S. Effect of pioglitazone on insulin resistance in fructose-drinking rats correlates with AGEs/RAGE inhibition and block of NADPH oxidase and NF kappa B activation. Eur. J. Pharmacol. 2010;629:153–158. doi: 10.1016/j.ejphar.2009.11.059.
    1. Ma N., Siegfried C., Kubota M., Huang J., Liu Y., Liu M., Dana B., Huang A., Beebe D., Yan H., et al. Expression Profiling of Ascorbic Acid-Related Transporters in Human and Mouse Eyes. Investig. Ophthalmol. Vis. Sci. 2016;57:3440–3450. doi: 10.1167/iovs.16-19162.
    1. Berthoud V.M., Beyer E.C. Oxidative stress, lens gap junctions, and cataracts. Antioxid. Redox Signal. 2009;11:339–353. doi: 10.1089/ars.2008.2119.
    1. Nita M., Grzybowski A. The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults. Oxid. Med. Cell. Longev. 2016;2016:3164734. doi: 10.1155/2016/3164734.
    1. Cheng P.W., Lin Y.T., Ho W.Y., Lu P.J., Chen H.H., Lai C.C., Sun G.C., Yeh T.C., Hsiao M., Tseng C.J., et al. Fructose induced neurogenic hypertension mediated by overactivation of p38 MAPK to impair insulin signaling transduction caused central insulin resistance. Free Radic. Biol. Med. 2017;112:298–307. doi: 10.1016/j.freeradbiomed.2017.07.022.
    1. Kojima M., Sun L., Hata I., Sakamoto Y., Sasaki H., Sasaki K. Efficacy of alpha-lipoic acid against diabetic cataract in rat. Jpn. J. Ophthalmol. 2007;51:10–13. doi: 10.1007/s10384-006-0384-3.
    1. Gul A., Rahman M.A., Hasnain S.N., Salim A., Simjee S.U. Could oxidative stress associate with age products in cataractogenesis? Curr. Eye Res. 2008;33:669–675. doi: 10.1080/02713680802250939.
    1. Wang F., Ma J., Han F., Guo X., Meng L., Sun Y., Jin C., Duan H., Li H., Peng Y. DL-3-n-butylphthalide delays the onset and progression of diabetic cataract by inhibiting oxidative stress in rat diabetic model. Sci. Rep. 2016;6:19396. doi: 10.1038/srep19396.
    1. Lim J.C., Perwick R.D., Li B., Donaldson P.J. Comparison of the expression and spatial localization of glucose transporters in the rat, bovine and human lens. Exp. Eye Res. 2017;161:193–204. doi: 10.1016/j.exer.2017.06.012.
    1. Shepherd P.R., Gibbs E.M., Wesslau C., Gould G.W., Kahn B.B. Human small intestine facilitative fructose/glucose transporter (GLUT5) is also present in insulin-responsive tissues and brain. Investigation of biochemical characteristics and translocation. Diabetes. 1992;41:1360–1365. doi: 10.2337/diab.41.10.1360.
    1. Chan C.Y., Guggenheim J.A., To C.H. Is active glucose transport present in bovine ciliary body epithelium? Am. J. Physiol. Cell Physiol. 2007;292:C1087–C1093. doi: 10.1152/ajpcell.00048.2006.
    1. Hashim Z., Zarina S. Advanced glycation end products in diabetic and non-diabetic human subjects suffering from cataract. Age. 2011;33:377–384. doi: 10.1007/s11357-010-9177-1.
    1. Elliott S.S., Keim N.L., Stern J.S., Teff K., Havel P.J. Fructose, weight gain, and the insulin resistance syndrome. Am. J. Clin. Nutr. 2002;76:911–922. doi: 10.1093/ajcn/76.5.911.
    1. Leibowitz A., Rehman A., Paradis P., Schiffrin E.L. Role of T Regulatory Lymphocytes in the Pathogenesis of High-Fructose Diet-Induced Metabolic Syndrome. Hypertension. 2013;61:1316–1321. doi: 10.1161/HYPERTENSIONAHA.111.203521.
    1. Conde S.V., da Silva T.N., Gonzalez C., Carmo M.M., Monteiro E.C., Guarino M.P. Chronic caffeine intake decreases circulating catecholamines and prevents diet-induced insulin resistance and hypertension in rats. Br. J. Nutr. 2012;107:86–95. doi: 10.1017/S0007114511002406.
    1. Chao E.C., Henry R.R. SGLT2 inhibition—A novel strategy for diabetes treatment. Nat. Rev. Drug Discov. 2010;9:551–559. doi: 10.1038/nrd3180.
    1. Chylack L.T., Jr., Wolfe J.K., Singer D.M., Leske M.C., Bullimore M.A., Bailey I.L., Friend J., McCarthy D., Wu S.Y. The Lens Opacities Classification System III. The Longitudinal Study of Cataract Study Group. Arch. Ophthalmol. 1993;111:831–836. doi: 10.1001/archopht.1993.01090060119035.
    1. Kilkenny C., Browne W., Cuthill I.C., Emerson M., Altman D.G. Animal research: Reporting in vivo experiments: The ARRIVE guidelines. Br. J. Pharmacol. 2010;160:1577–1579. doi: 10.1111/j.1476-5381.2010.00872.x.
    1. McGrath J.C., Lilley E. Implementing guidelines on reporting research using animals (ARRIVE etc.): New requirements for publication in BJP. Br. J. Pharmacol. 2015;172:3189–3193. doi: 10.1111/bph.12955.
    1. Curtis M.J., Bond R.A., Spina D., Ahluwalia A., Alexander S.P., Giembycz M.A., Gilchrist A., Hoyer D., Insel P.A., Izzo A.A., et al. Experimental design and analysis and their reporting: New guidance for publication in BJP. Br. J. Pharmacol. 2015;172:3461–3471. doi: 10.1111/bph.12856.

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