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Effect of protein glycation by methylglyoxal on pancreatic beta cell function
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Tym, Amy (2014) Effect of protein glycation by methylglyoxal on pancreatic beta cell function. PhD thesis, University of Warwick.
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WRAP_THESIS_Tym_2014.pdf - Submitted Version - Requires a PDF viewer. Download (3998Kb) | Preview |
Official URL: http://webcat.warwick.ac.uk/record=b2725034~S1
Abstract
Methylglyoxal is a physiological dicarbonyl metabolite and potent argininedirected glycating agent. It often modifies proteins at functional sites producing loss of positive charge, structural distortion and inactivation. Plasma methylglyoxal is increased in hyperglycaemia associated with diabetes and is linked to the development of vascular complications of diabetes – particularly nephropathy, retinopathy and neuropathy. The effects of dicarbonyl glycation on beta cells and involvement in early stage dysfunction and development of type 2 diabetes mellitus are not known. The aim of this project was to investigate the effect of dicarbonyl protein glycation on beta cell function and related involvement in the development of diabetes. Studies were performed in an in vitro model of beta cell dysfunction - MIN6 insulinoma cells incubated under low and high glucose concentrations, and in a pre-clinical in vivo model of decline of glucose tolerance preceding development of type 2 diabetes - high fat diet-induced insulin resistant mice. Dicarbonyl metabolism and protein damage by glycation and oxidation were studied by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Localisation of methylglyoxal glycation adducts within the pancreas were visualised by immunostaining. Interactions between the extracellular matrix protein, collagen IV, and MIN6 cells in vitro were investigated and impairments in adhesion were assessed following glycation with methylglyoxal. Impairments in adhesion of MIN6 cells to methylglyoxal-glycated collagen IV were assessed using atomic force microscopy force spectroscopy.
The results show that MIN6 cells were resistant to accumulation of methylglyoxal when incubated in high glucose concentration although the flux of methylglyoxal was increased 41%. Glycation of collagen IV by methylglyoxal impairs binding to MIN6 cells in vitro resulting in a 91% decrease in the energy necessary to detach cells from the extracellular matrix protein. In high fat diet fed mice the concentration of methylglyoxal in the pancreas was increased. Visualisation of MG-H1 adduct residues in the pancreas showed they were predominantly on the extracellular matrix.
In conclusion, protein glycation by methylglyoxal occurs in MIN6 cells in vitro and in the mouse pancreas in vivo. Although the methylglyoxal concentration in the pancreas of high fat diet fed, insulin resistant mice was increased, the lack of a concurrent increase in methylglyoxal protein glycation adducts suggests there may be increased turnover of methylglyoxal-modified proteins. Impairment of beta cell attachment to the extracellular matrix protein, collagen IV, by methylglyoxal and increased protein turnover stimulated by an increased rate of methylglyoxal
glycation may impair beta cell function in pre-diabetes in vivo. Glycation by methylglyoxal may contribute to beta cell glucotoxicity and dysfunction with progression to type 2 diabetes mellitus.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QP Physiology | ||||
Library of Congress Subject Headings (LCSH): | Glycosylation, Proteins, Pancreatic beta cells, Non-insulin-dependent diabetes -- Pathophysiology, Extracellular matrix proteins | ||||
Official Date: | February 2014 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences ; Warwick Medical School | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Thornalley, Paul J.; Squires, Paul | ||||
Sponsors: | Biotechnology and Biological Sciences Research Council (Great Britain) | ||||
Extent: | 231 leaves : charts. | ||||
Language: | eng |
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