May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Proteomic Analysis of Advanced Glycation Endproduct (AGE)-Modified Proteins in Müller Cells During High Glucose Exposure: Implications for Macroglial Dysfunction During Diabetic Retinopathy
Author Affiliations & Notes
  • R. S. Pringle
    Centre for Vision Science, Queens University Belfast, Belfast, United Kingdom
  • R. Nagai
    Kumamoto University, Kumamoto, Japan
  • J. Robson
    School of Biological and Biomedical Science, Durham University, Durham, United Kingdom
  • J. W. Curry
    Centre for Vision Science, Queens University Belfast, Belfast, United Kingdom
  • A. Stitt
    Centre for Vision Science, Queens University Belfast, Belfast, United Kingdom
  • Footnotes
    Commercial Relationships  R.S. Pringle, None; R. Nagai, None; J. Robson, None; J.W. Curry, None; A. Stitt, None.
  • Footnotes
    Support  DEL
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 1336. doi:https://doi.org/
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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)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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).

Conclusions: : 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.

Keywords: diabetic retinopathy • glia • proteomics 
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