Dicarbonyl glycation - particularly glycation by methylglyoxal (MG) - is an important type of spontaneous damage to the proteome. Proteins that are susceptible to glycation by MG with consequent functional impairment are called collectively the dicarbonyl proteome. Methylglyoxal-derived advanced glycation endproducts (AGEs) are abundant in vascular tissues and are thought to contribute to the development of vascular complications of diabetes. Dicarbonyl stress is the imbalance of reactive dicarbonyls and anti-glycation defences leading to increased AGEs. Previous studies have shown that the concentration of MG is increased in human microvascular endothelial cells in vitro incubated in a model of hyperglycaemia. The glyoxalase system is an anti-glycation defence in all cells that metabolises MG and thereby suppresses MG-mediated protein damage. Overexpression of Glo1 decreased the intracellular levels of MG. Recently it has been reported that primary aortic endothelial cells incubated under high glucose concentration in vitro showed decreased activity of Glo1. The aim of this investigation was to improve the understanding of protein damage in vascular disease in diabetes, -focusing on protein damage by MG in vascular endothelial cells in hyperglycaemia. The effect of high glucose concentrations on formation and metabolism of methylglyoxal was studied in human vascular endothelial cells in vitro. The ability of a Glo1 inducer, resveratrol (RSV), to counter dicarbonyl stress in human endothelial cells in vitro was also assessed. In vivo effects of Glo1 deficiency and over expression on dicarbonyl glycation were also studied in mouse model of diabetic vascular disease. The results show that methylglyoxal concentration is increased by 2 - 3 fold in culture medium and ca. 109% inside human vascular endothelial cells during culture in model hyperglycaemia which is returned to normal by RSV treatment. In addition, the flux of formation of D-lactate, the terminal product of MG metabolism by the glyoxalase system, is increased by 30% in endothelial cell cultured in model hyperglycaemia in vitro is also prevented by RSV. Furthermore, cellular activity of glyoxalase 1 and protein content is decreased by 20 - 30% with high glucose culture due to increased ubiquitination of Glo1 in human vascular endothelial cells in vitro suggesting increased proteolysis of Glo1. RSV also protected the decrease in Glo1 activity in these cells. However the increased formation of AGEs free adducts observed in high glucose conditions were not corrected with RSV but the level of MG-damaged proteins in cells was protected. Disturbance of methylglyoxal metabolism in an experimental model of diabetic vascular disease, Glo1 deficient mice at 20 weeks did not show any increase in dicarbonyl glycation of aortal collagen. However at 35 weeks the major MG and glyoxal derived AGE - MG-H1 and CML - were significantly increased possibly due to deterioration in metabolic resistance to dicarbonyl stress with age or/and decreased turnover in vascular collagen. Overexpression of Glo1 restricted to sites of the proendothelin promoter (endothelial cells and smooth muscle cells) in mice was unable to decrease aortal dicarbonyl glycation and atherosclerosis. This study reveals that the defence against dicarbonyl glycation is decreased in endothelial cells in high glucose in vitro and the flux of formation of methylglyoxal is increased. Induction of Glo1 expression may contribute to health benefits of RSV and Glo1 inducers may protect against the development of vascular complications in diabetes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606140 |
| Date | January 2013 |
| Creators | Irshad, Zehra |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/60536/ |
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