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Saeed Yadranji-Aghdam, Christine Sorenson, Nader Sheibani; High Glucose and Diabetes Modulate Cellular Proteasome Function: Implications in the Pathogenesis of Diabetes Complications. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1153.
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The precise link between hyperglycemia and its deleterious effects on the retinal and kidney microvasculature, and more specifically loss of perivascular supporting cells including smooth muscle cell/pericytes (SMC/PC), are not completely understood. We hypothesized that differential cellular proteosome activity contributes to sensitivity of PC to high glucose-mediated oxidative stress and vascular rarefaction.
Retinal endothelial cells (EC), PC, and choroidal EC were prepared and cultured as previously described. NIH3T3 and 661W cells were obtained from ATCC. These cells were cultured under various glucose conditions for 5 days prior to any analysis. Wild type or Akita/+ mice with different duration of diabetes were obtained from Jackson laboratories. For in vitro proteasome peptidase assays, the cells or tissue lysates (50µl of 1µg/µl) per assay were loaded into each well of a dark 96-well microplate and were probed with 100µM of each of the synthetic fluorogenic peptide substrates for caspase-like, trypsin-like, and chymotrypsin- and calpain-like protease activity. The expression levels and localization of various proteins were analyzed by Western blotting and immunofluorescence, respectively. The mRNA expression levels were determined by quantitative real time PCR.
Here we show that retinal EC have significantly higher proteasome peptidase activity compared to PC. High glucose treatment (HGT) increased the level of total ubiquitin-conjugated proteins in cultured retinal PC and EC, but not photoreceptor cells. In addition, in vitro proteasome activity assays showed significant impairment of proteasome chymotrypsin-like peptidase activity in PC, but not EC. The HGT-mediated rise in ubiquitination was attenuated by treatment with N-acetylcysteine. Furthermore, HGT increased the half life and protein levels of the members of the 11S regulatory subunits of mammalian proteasomes (PA28-a/-b proteins) in retinal PC, but not EC or photoreceptor cells. The PA28-a/-b protein levels were also higher in the retina and kidney glomeruli of diabetic mice.
Together our results demonstrate that high glucose has direct biological effects on cellular proteasome function in a cell type-specific manner, and this modulation might be protective against cellular stress or damage induced by high glucose.
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