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