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Title: An investigation into the mechanisms of lipotoxicity in rodent and human-derived β-cell lines
Author: Maloney, P.
ISNI:       0000 0004 8498 2223
Awarding Body: University of Exeter
Current Institution: University of Exeter
Date of Award: 2019
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Background and aims: In vitro studies with rodent beta cells suggest that individual free fatty acids (FFA) can exert differential effects such that long-chain saturated fatty acids (LC-SFA) promote toxicity while their monounsaturated counterparts (e.g. C18:1) are benign. This is potentially important, if also true in humans, because patients with type 2 diabetes often display elevated circulating FFA. Furthermore, elevated levels of odd chain SFA (C15:0, C17:0) have been associated with a reduced prevalence of diabetes, but their impact on beta cell viability has not been assessed. The investigations detailed within this thesis have characterised the effects of various FFA on beta cell viability. Further, the subcellular distribution of long-chain fatty acids (LC-FFA) has been characterised to elucidate the underlying mechanisms of lipotoxicity in the human EndoC-βH1 and the rat INS-1 beta cell lines. Methods: Cells were exposed to a range of LC-FFA for increasing periods and viability was subsequently assessed using either vital dye staining or flow cytometry. The distribution of LC-FFA within cells was studied using a fluorescent palmitate analogue, BODIPY FL C16, and Transmission Electron Microscopy (TEM). Golgi co-localisation was determined with the aid of Golgi-RFP. The oxygen consumption rate (OCR) of cells was measured using a Seahorse XF96e analyser. Results: Exposure of INS-1 cells to C15:0, C16:0, C17:0 and C18:0 caused a dose-dependent loss of viability over 24h, which was completely attenuated with the co-incubation of C18:1. Conversely, exposure of EndoC-βH1 cells to C15:0, C16:0, C17:0 and C18:0 did not cause a loss in viability, even at concentrations up to 1mM, and for exposure periods of 72hrs. Furthermore, EndoC-βH1 cells were resistant to the cytotoxic effects of C16:0 (0.5mM) and glucose (20mM) combined, a phenomenon previously observed in rodent beta cells. Interestingly, the stearoyl-CoA desaturase (SCD-1) inhibitor 10,12-CLA, and the V-ATPase inhibitor, bafilomycin, both caused EndoC-βH1 cell death. The long-chain monounsaturated fatty acid (LC-MUFA) C18:1 also caused a modest increase in EndoC-βH1 cell death relative to control, although cell death was not observed in those cells treated with C16:1. In the INS-1 cell line, exposing cells to both C16:0, C18:1 and the fluorescent tracer BODIPY FL C16, caused C16:0 to become distributed in a punctate manner throughout the cytosol, a feature not observed when INS-1 cells were solely treated with C16:0 and BODIPY FL C16. Treating INS-1 cells with unlabelled C16:0 caused the ER to appear dilated. Moreover, C16:0 was found to accumulate at the Golgi apparatus. In contrast, C18:1 did not cause swelling of the Golgi apparatus; it did not cause alterations to ER morphology and C18:1 was routed to the mitochondria for oxidation. To what extent C18:1 is oxidised in INS-1 cells, however, remains unclear. Strikingly, C16:0-induced ER dilation and swelling of the Golgi apparatus was not observed in the presence of C18:1. In the EndoC-βH1 cell line, similarly to the INS-1 cell line when treated with C18:1, C16:0 did not localise to the Golgi, was distributed in a punctate manner throughout the cytosol, and did not cause the ER to become dilated. Furthermore, cytosolic puncta were observed in EndoC-βH1 cells treated with C16:0 and BODIPY FL C16¬ in combination with C17:0, C19:0, and C18:1. Conclusion: Differences observed in the viability profile of LC-FFA in human compared to rodent β-cells may be due to differential routing of lipids. Further research is required to determine whether lipotoxicity occurs in human pancreatic β-cells in the pathophysiology of T2D.
Supervisor: Morgan, N. G. ; Welters, H. J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available