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R. S. Pringle, A. K. Berner, H. Zong, R. Nagai, A. limb, J. W. Curry, A. W. Stitt; Proteomic Analysis of Advanced Glycation End-Product (Age) Modified Proteins in High Glucose-Exposed Retinal Müller Glia: Evidence for Age-Mediated Cell Dysfunction During Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4992.
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© ARVO (1962-2015); The Authors (2016-present)
Müller glia show enhanced glycolysis during diabetes and a consequence of this metabolism is cytoplasmic elevations in the reactive dicarbonyl methylglyoxal (MGO). MGO is a well-defined AGE precursor and many cells possess a glyoxalase enzyme system (GLO1 and GLO2) that detoxify MGO and limit AGE accumulation. Using a proteomic screening approach for MGO-derived AGEs we have sought to establish links to AGEs and retinal Müller glia dysfunction during diabetes. We have also over-expressed GLO-1 to determine if this could limit AGE-mediated protein modification in high glucose-exposed Müller glia.
Human Müller cells (MIO-M1) were exposed to normal (5mM) or high glucose (25mM and 45mM) (HG) for 10 days. The same treatment was performed on MIO-M1 cells stably transfected to over-express GLO1. Proteins were separated using 2-D gel electrophoresis (2DE) (pH 4-7) and changes to the proteome identified using Progenesis software (n=3). Western analysis of 2D gels (n=3) identified proteins modified by MGO. Proteins shown to have AGE modifications were identified by MALDI-TOF and MS-MS mass spectroscopy analysis and Mascot search algorithms. The effects of HG on Müller glia (wild-type and GLO1 over-expressing) where further analysed for cell cycle response, apoptosis and inflammatory cytokine expression.
HG induced significant AGE protein adducts in MIO-M1, whilst over-expression of GLO1 reduced adduct formation. We identified ~70 proteins modified by MGO-derived AGEs and these included major structural proteins as well as key antioxidant/metabolism enzymes, heat shock proteins and proteins related to apoptosis. GLO1 over-expression reduced high glucose mediated AGE protein adducts. Apoptosis of MIO-M1 was not significantly altered between treatments or transfected cells. Following HG exposure for both wild-type and Glo1 over-expression MIO-M1 upregulated inflammatory cytokines mRNA (including IL-1β,IL-8, and VEGF) (P<0.01). Glo1 over-expression attenuated HG-mediated glutamate release from MIO-M1 (P<0.005)
MGO-derived AGEs are abundant in Müller glia exposed to high glucose. Adduct formation on key proteins could significantly alter their function. Protection from MGO formation and subsequent AGE modification by enhancing GLO1 detoxification partially protects Müller glia from diabetes-related dysfunction.
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