Type 2 diabetes (T2D) is a growing health-care and economic burden. Obesity is a risk factor for developing T2D, but the underlying molecular mechanisms are not well understood. However, mechanisms such as lipotoxicity, endoplasmic reticulum stress and inflammation are becoming increasingly well-recognised in obesity, and may underlie the development and progression of T2D. A central player in these mechanisms is Protein Kinase R (PKR), proposed to have a role within nutrient- and pathogen-sensing pathways, and is activated by ER stress and lipotoxicity. A small molecule inhibitor Compound-16, adenoviral vectors and RNAi techniques in BRIN-BD11 rodent pancreatic β-cells, were used to demonstrate that PKR knockdown affords significant protection against palmitate-induced cell death. Furthermore, PKR knockdown potentiates palmitoleate cytoprotection during lipotoxicity, suggesting the cytotoxic and cytoprotective actions of long-chain fatty acid species may function via the PKR signalling pathway. The use of a novel 1.1B4 human pancreatic β-cell line has shown that important differences exist between human and rodent cell responses to fatty acids in vitro. In 1.1B4 cells, long-chain saturated and monounsaturated fatty acids do not provide increasing protection as their chain-length increases, in contrast to rodent cell models. Furthermore, methyl-saturated fatty acid species are well tolerated, and methyl-monounsaturated fatty acids are cytoprotective to 1.1B4 β-cells. TXNIP overexpression in an INS-TXNIP β-cell model has a proapoptotic role in conditions of glucotoxicity, but not glucolipotoxicity. Furthermore, in this cell model, succinate is cytoprotective against glucotoxicity, but not glucolipotoxicity. By contrast in 1.1B4 β-cells, succinate significantly protects against apoptosis induced by both glucotoxic and glucolipotoxic conditions. Chronic inflammation has been implicated in the development and progression of T2D. At the centre of this response is the pro-inflammatory cytokine IL-1β. The cellular origin of IL-1β is unclear, but IL-1β secretion has been linked to activation of the NLRP3 inflammasome, recently implicated in pancreatic β-cell death in T2D. Results suggest that IL-1β is secreted by INS-TXNIP and 1.1B4 pancreatic β-cells under lipotoxic conditions, thus offering a potential role for targeted IL-1β therapy in T2D.