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R. S. Pringle, R. Nagai, J. Robson, J. W. Curry, A. Stitt; Proteomic Analysis of Advanced Glycation Endproduct (AGE)-Modified Proteins in Müller Cells During High Glucose Exposure: Implications for Macroglial Dysfunction During Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1336. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal Müller cells demonstrate increased glycolytic metabolism which precipitates enhanced intracellular levels of the dicarbonyl methylglyoxal (MGO). MGO is a precursor for AGE-adduct formation and this pathway could make an important contribution to macroglial damage in early diabetic retinopathy. This study used a proteomic approach to identify MGO-modified proteins in Müller cells exposed to high glucose.
Human Müller cells (MIO-M1; a gift from Dr Astrid Limb, Institute of Ophthalmology, London) were exposed to normal (5mM) and high glucose (25mM) conditions for 10 days. Proteins were separated using 2-D gel electrophoresis (2DE) (pH 4-7) and changes to the proteome identified using Progenesis software. 2DE western analysis (n=3) identified significantly greater numbers of MGO-immunoreactive proteins from Müller cells exposed to high glucose compared to normal conditions. Selected proteins were identified by MALDI-TOF mass spectroscopy analysis and Mascot search algorithms. MGO-modified proteins were subsequently assessed in Müller cells using "conventional" western blotting and immunocytochemical analyses.
136 MGO modified proteins were identified in cells grown in high glucose compared to 65 in controls. Identified target proteins (p<0.05) from high glucose-exposed cells included α/β-tubulin, β-actin, and 3 serum albumin proteins. Two forms of a human insulin inhibitor, α-2-HS-glycoprotein, linked to insulin resistance, were identified. Protein disulphide isomerase, an enzyme in the endoplasmic reticulum that catalyzes protein folding was also MGO-modified. A further 38 identified proteins were attributed to high glucose (upregulated: 14-3-3 Protein theta, ATPase, Ubiquitin activating enzyme E1; down-regulated: cathepsin B/ D, enolase 1).
Short-term high glucose exposure leads to significant formation of MGO-derived AGEs in the Müller cell proteome. AGEs are known to have deleterious effects on protein structure and function; we have identified modifications that have important implications for retinal macroglial dysfunction in diabetes.
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