Toll-like receptor agonist induced changes in clonal rat BRIN-BD11 b-cell insulin secretion and signal transduction Aoife Kiely, Aisling Robinson, Neville H McClenaghan 1 , Peter R Flatt 1 and Philip Newsholme School of Biomolecular and Biomedical Sciences, Conway Institute and Health Sciences Centre, UCD Dublin, Belfield, Dublin 4, Ireland 1 School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK (Correspondence should be addressed to P Newsholme; Email: philip.newsholme@ucd.ie) Abstract Evidence for involvement of toll-like receptors (TLRs) (e.g. TLR4 and TLR2, whose agonists include lipopoly- saccharides (LPS) and saturated fatty acids) in altered patterns of signalling in adipose, liver and muscle from animal models of insulin resistance and obesity has been published. We have now extended this area of research and have determined the effects of LPS on cell viability, insulin secretion, insulin signalling and metabolism in a clonal b-cell line. BRIN-BD11 b-cells were treated for 24 h with increasing concentrations of LPS. Chronic (24 h) and acute (20 min) insulin secretion, insulin content and parameters of cell metabolism and insulin signalling were determined. Incu- bation of BRIN-BD11 cells for 24 h in the presence of increasing concentrations of the TLR4 ligand LPS signi- ficantly decreased chronic (24 h) insulin secretion from 1 . 09G0 . 19 to 0 . 76G0 . 18 mg insulin/mg protein in the presence of 100 ng/ml LPS (P!0 . 05). There was no change in acute (20 min) stimulated insulin secretion or insulin con- tent. Cell metabolism was not changed. Insulin receptor-b (IRb) expression levels were increased significantly from 1G0 . 52 to 8 . 6G1 . 83 units (P!0 . 01), whereas calcineurin activity and Akt phosphorylation were significantly (P!0 . 01 and P!0 . 05 respectively) reduced in response to 24 h incubation in the presence of LPS. There was no change in IR substrate-1 protein expression or phosphorylation after 24 h. Further incubation for 24 h in the absence of LPS resulted in the recovery of chronic insulin secretion. The negative b-cell effects of LPS may contribute to hyperglycaemia in vivo . Journal of Endocrinology (2009) 202, 365–373 Introduction Toll-like receptors (TLRs) are a family of evolutionarily conserved receptors primarily involved in regulation of the immune system by sensing a range of chemicals produced by pathogens such as bacteria and viruses and activating immune cell responses (O’Neill 2006). TLR signalling has a number of effects on the activities of antigen presenting cells, including production of inflammatory cytokines and up-regulation of MHC products. TLR expression has been demonstrated on cells such as macrophages and dendritic cells (Atkinson 2008). However, the expression has also been reported on numerous other cells throughout the body, including those from various tissues and organs and some cell lines (Vives-Pi et al. 2003, Tsukumo et al. 2007). Indeed, TLR4, TLR2 and the leucine-rich repeat MD-2 proteins have been detected in islet cells, thus suggesting that b-cells express a functional lipopolysaccharide (LPS) receptor (Vives-Pi et al. 2003). Evidence for involvement of innate immune product receptors, e.g. TLRs, in insulin resistance has been recently published (Tsukumo et al. 2007). Notably type 2 diabetes has been associated with chronic low-grade inflammation (Creely et al. 2007) in addition to the well-defined tissue insulin resistance (which is the result of changes in insulin receptor (IR) coupled signal transduction pathways in muscle, adipose tissue and liver, Tsukumo et al. 2007). As well as innate immune products such as LPS, adaptive immune factors such as pro-inflammatory cytokines (such as tumour necrosis factor-a (TNF-a) and interleukin-1b (IL-1b)) can alter patterns of insulin signalling in these target tissues, resulting in resistance to the action of insulin (Tilg & Moschen 2008). Recent work has implicated fatty acids as agonists for TLR4 and TLR2 and thus established a possible connection between altered patterns of insulin signalling in animal models of insulin resistance and obesity (Lee et al. 2003). In contrast to saturated fatty acids, polyunsaturated fatty acids inhibit this pathway (Poltorak et al. 1998, Lee et al. 2003, Tsukumo et al. 2007). Loss of function mutations in the TLR4 receptor had a beneficial effect on the insulin signalling pathways in adipose, muscle and liver tissues in mice and improved insulin action (Tsukumo et al. 2007). Human adipocytes were demonstrated to have inducible TLR4 and TLR2 pathways further supporting the role that adipose tissue may play in the regulation of inflammation (Vitseva et al. 2008). 365 Journal of Endocrinology (2009) 202, 365–373 DOI: 10.1677/JOE-09-0160 0022–0795/09/0202–365 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 06/06/2020 11:18:43AM via free access