Biophysical and biochemical effects and distribution of fatty acids in pancreatic beta cells and microvascular endothelial cells
Date: 25 March 2019
University of Exeter
PhD in Medical Studies
The incidences of obesity and type 2 diabetes and their complications are increasing globally. The presence of elevated circulating free fatty acids has been associated with the initial dysfunction of pancreatic beta cells and microvascular endothelial cells followed later by their demise. The aim of this thesis was to investigate the ...
The incidences of obesity and type 2 diabetes and their complications are increasing globally. The presence of elevated circulating free fatty acids has been associated with the initial dysfunction of pancreatic beta cells and microvascular endothelial cells followed later by their demise. The aim of this thesis was to investigate the mechanisms by which demise occurs, and how it may be prevented. Palmitate, a saturated fatty acid, caused cell death in both INS-1 beta cells and HCMec/D3 microvascular cells, whereas the unsaturated fatty acid oleic acid did not cause cell death, and also protected against palmitate-induced toxicity. Etomoxir, the mitochondrial CPT1 inhibitor did not rescue INS-1 or HCMec/D3 cells from palmitate-induced toxicity suggesting that palmitate-induced toxicity does not occur via entry into the mitochondria. Cells were exposed to 2-bromopalmitate, a non-metabolisable fatty acid used to reduce the pool of cytoplasmic CoA, to determine whether palmitate-induced toxicity might be mediated by its ability to be activated. Pre-incubation with 2-bromopalmitate in INS-1 cells significantly prevented palmitate-induced cell death. These data suggest that the activation of palmitate with CoA might mediate cell death. Cell cycle analysis found that neither oleic acid nor palmitate caused an increase or decrease in cell proliferation in both INS-1 and HCMec/D3 cells. The data suggest that the mechanism of oleic acid-induced cytoprotection might not be via a pro-proliferative mechanism. INS-1 cells were imaged using spontaneous Raman microspectroscopy after 24-hour exposure to esterified and non-esterified fatty acids. Uni- and multi-variate analysis and spectral decomposition were carried out using a methodology optimised and validated which is presented in this thesis. The aim was to quantify changes, if any, in lipid disposition: distribution, intensity (as a measure of concentration) and composition after exogenous exposure to these fatty acids. Exposure to 0.125 mM palmitate showed a significant decrease in the percentage of lipid within the cells and a corresponding increase in the intensity of this lipid. This suggests that palmitate, alone, might be shuttled into lipid droplets. This was not observed when the cells were exposed to oleic acid, whereby an increase in the intensity of lipid was observed even though no significant change was observed in the percentage of lipid within the cells. When palmitate and oleic acid were combined, the composition of the lipid droplets changed such that the levels of palmitate decreased and the levels of oleic acid increased. These data suggest that oleic acid does not shuttle palmitate into lipid droplets. These data do not support the hypothesis that oleic acid protects against palmitate-induced cytotoxicity by shuttling palmitate into lipid droplets. The methyl esters of palmitate and oleic acid were employed to determine whether they would affect lipid disposition. No change in lipid distribution or intensity was observed when the cells were exposed to these fatty acids, validating the requirement for the free carboxyl oxygen for the covalent binding to glycerol for the formation of lipid droplets. These data also suggest that INS-1 cells cannot de-esterify esterified fatty acids.
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