AbstractThe overproduction of reactive oxygen species (ROS), leading to oxidative stress, is central to the induction of hyperglycaemia-mediated endothelial dysfunction and the onset of diabetes-related cardiovascular disease (CVD). In vitro treatment with high glucose is known to increase ROS production in endothelial cells. However, the comparative effect of intermittent-high glucose on ROS production in endothelial cells is yet to be fully established. Moreover, the source(s) and mechanism(s) of high glucose-induced superoxide (the primary ROS) overproduction remain unclear.
The first aim of this thesis was to utilise an in vitro model of high and intermittenthigh glucose to measure the comparative impact of treatments on ROS production, cell viability/toxicity, and cell proteome, in human umbilical endothelial cells (HUVECs). Secondly, the impact of high and intermittent-high glucose on bioenergetic function in HUVECs was assessed, using extracellular flux analysis.
Lastly, the impact of improved glucose control on oxidative stress was retrospectively determined using data from a clinical study of T2DM patients receiving insulin pump therapy.
High glucose significantly increased both general and mitochondrial superoxide generation in HUVECs, whereas intermittent-high glucose did not. High glucose also increased the total NAD+/NADH ratio and appeared to elicit changes in the cell proteome, whilst not significantly affecting cell survival. High and intermittent-high glucose significantly decreased the OCR/ECAR ratio in HUVECs, driven primarilyby an increase in glycolytic metabolism. Glucose treatments had little impact on the mitochondrial respiratory function in HUVECs, due to the inherent lack of reserve respiratory capacity in the cells. Finally, better glucose control was found to correlate with decreased oxidised LDL cholesterol in patients with diabetes – an effect that was augmented by statin use.
In conclusion, it is apparent that high glucose concentrations, rather than intermittent periods of high/low glucose, are responsible for increased superoxide production in HUVECs. Furthermore, treatment with high glucose within the pathophysiologically relevant range in diabetes can induce changes to both the cellular proteome and pyridine nucleotide metabolism. Although exposure to both high and intermittenthigh glucose alter HUVEC bioenergetics, the precise nature of the link between glucose metabolism and increased mitochondrial superoxide production in endothelial cells remains to be elucidated. However, increased mitochondrial substrate oxidation under unfavourable conditions remains a feasible hypothesis.
Lastly, better long-term glucose control correlating with lower ox-LDL suggests that the extent and duration of an individual’s glucose excursions may be very much relevant to the lasting effects of diabetes-related complications.
|Date of Award||31 Jul 2015|
|Supervisor||Ian Megson (Supervisor), Sandra MacRury (Supervisor) & Mary Doherty (Supervisor)|