Katy Louise Hayward
Lipotoxicity and the role of translationally controlled tumour protein (TCTP) in pancreatic ?-cell survival
Hayward, Katy Louise
Introduction: Diabetes affects more than 346 million individuals worldwide. Some 90% of diabetics have type 2 diabetes mellitus, which is frequently associated with obesity and hyperlipidemia as well as hyperglycaemia. There is ample evidence that fatty acids become toxic (lipotoxicity) when present at elevated concentrations for prolonged periods of time, although mechanisms are still not fully elucidated. Current diabetic medications do not tackle the underlying issue of β-cell death. New therapeutic strategies to more effectively combat and early deterioration of the β-cell mass and function due to lipotoxicity are thus required. Translationally Controlled Tumour Protein (TCTP) has been identified in a wide range of eukaryotic organisms and linked to many diverse cellular processes including acting as an anti-apoptotic protein. Ideally human islets of Langerhans would be used to investigate diabetes however they are not practical to use due to only being acquirable from cadavers. Rodent islets are more readily available however keeping the rodents require a lot of money, time and a license to ensure they are being cared for correctly. Monolayer cell lines have been created for the use in basic research which can be grown in a suspension forcing the cells to attach to each other forming 3D structures commonly referred to as pseudo-islets, have shown some promising results.
Aims: (1) establishing an imaging-based assay for analysing characteristic changes in pancreatic β-cells lipotoxicity, (2) investigate the effect of TCTP in connection with lipotoxicity and (3) investigate possible alternatives to using cultured monolayer cells or isolated islets of Langerhans for diabetes research.
Methods: MIN6, INS-1, HIT-T15, alpha TC 1 clone 6 cells and Hans-Wistar rat islets were incubated with were incubated with forskolin, exendin-4, thapsigargin, palmitate and oleate/palmitate (1:1) mix under stimulatory glucose conditions for 8, 24 or 48h to investigate lipid accumulation and/or protein changes. Lipid accumulation and cell death was investigated using ImageXpress 5000a and confocal microscopy. Changes in protein expression were investigated using immunoblots using a mouse monoclonal anti-TCTP antibody. Equal amount of protein was loaded into each lane and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) were used as internal controls. Changes in TCTP mRNA expression levels were investigated using TaqMan® with real-time PCR. Pancreatic spheroid formation was investigated using MIN6 and INS-1cells via three methods: agarose overlay, hanging drop/methyl cellulose, and confocal microscopy.
Conclusions: Lipid accumulation could successfully be tracked in monolayer cells and islets of Langerhans. Proteomic and biochemical approaches revealed that TCTP level is regulated by glucose, palmitate and exendin-4. Regulation of TCTP by glucose and exendin-4 is cyto-protective. In contrast, high concentration of palmitate causes cell stress, reduction in TCTP protein level and consequently reduced cell viability. This study demonstrated that the cytotoxic effects of palmitate are at least in part mediated by the inhibition of the production of the anti-apoptotic protein TCTP which is reversed with the addition of exendin-4. In contrast, TCTP mRNA was found to be elevated in response to high level of fatty acids both in MIN6 cells and Zucker fatty rat islets of Langerhans suggesting that β-cells try to compensate for the reduced translation of TCTP. The investigation in to an alternative method for spheroid creation failed to produce complete spheroids but a number of areas for future improvements were identified.
Hayward, K. L. Lipotoxicity and the role of translationally controlled tumour protein (TCTP) in pancreatic ?-cell survival. (Thesis). University of the West of England
|Keywords||TCTP, translationally controlled tumour protein, type 2 diabetes, lipotoxicity, palmitate|
|Additional Information||Corporate Creators : AstraZeneca|