Proceedings of The Physiological Society

Future Physiology (Leeds, UK) (2017) Proc Physiol Soc 39, PC38

Poster Communications

A20 regulation of pancreatic β-cell survival

J. McLaughlin1

1. Center for Stratified Medicine, Ulster University, Derry/Londonderry, Derry, United Kingdom.

Background: Apoptosis of the pancreatic β-cell is central to the pathogenesis of type 1 diabetes and results in an absolute insulin requirement that is replaced via exogenous sources of the hormone. The path to β-cell apoptosis in type 1 diabetes is complex, but in part, is driven by NF-κB mediated inflammation. A20 is an important endogenous cytoplasmic protein that negatively regulates NF-κB inflammation and apoptotic processes and is also the mostly highly regulated anti-apoptotic gene in the β-cell [1]. The objective of this study was to establish the effect of hyperglycaemia on the expression of A20, which is said to undergo glycosylation-driven proteasomal degradation under hyperglycaemic conditions [2]. Furthermore, we sought to establish if inhibition of glycosylation would prevent NF-κB activation and promote β-cell survival. Methods: BRIN-BD11 cells (rat pancreatic β-cell line) were cultured in RMPI containing 5 mM D-Glucose (normoglycaemic), or 25 mM D-Glucose (hyperglycaemic). TNFα rapidly and transiently induced A20 expression and cells were therefore challenged with 100 ng/ml TNFα for 0-24h. Comparisons of A20 expression between cells culture under normoglycaemic and hyperglycaemic conditions were conducted at the mRNA (qPCR) and protein level (Western Blot). NF-κB activation was also assessed in nuclear extracts using a NF-κB activity ELISA (Abcam) in the presence or absence of 10 μM DON, an inhibitor of glycosylation. All data are presented as the means ± SEM for a given number of observations (n). Student's t-tests were considered to be significant if p<0.05. Results: A20 mRNA expression was significantly (p<0.001, n=6) lower in cells cultured under hyperglycaemic conditions when compared with cells cultured in normoglycaemic conditions. This was consistent regardless of whether expression was measured under basal unstimulated conditions, or in the presence of TNFα. A total lack of A20 protein was observed under hyperglycaemic conditions when cells were treated with TNFα. NF-κB activation was found to be significantly (p<0.001, n=4) upregulated under hyperglycaemic conditions when cells were treated with TNFα, and this level of activation was significantly (p<0.01, n=4) reduced upon inhibition of glycosylation. Conclusion: Hyperglycaemia significantly reduces A20 expression, which is associated with heightened activation of NF-κB. Findings from this study support the assumption that the discovery of pharmacological treatments preventing the degradation of A20 could assist in the treatment of type 1 diabetes. Prevention of A20 degradation may limit β-cell apoptosis and the progression of type 1 diabetes.

Where applicable, experiments conform with Society ethical requirements