Acute effects of nutrient stimuli on pancreatic beta-cell function are widely reported; however, the chronic effects of insulinotropic amino acids, such as L-alanine, on pancreatic beta-cell function and integrity are unknown. In the present study, the effects of prolonged exposure (24 h) to the amino acid L-alanine on insulin secretory function, gene expression and pro-inflammatory cytokineinduced apoptosis were studied using clonal BRIN-BDII cells. Expression profiling of BRIN-BD I I cells chronically exposed to L-alanine was performed using oligonucleotide microarray analysis. The effect of alanine, the NOS (inducible nitric oxide synthase) inhibitor NMA (N-G -methyl-L-arginine acetate) or the NOS and NADPH oxidase inhibitor DPI (diphenylene iodonium) on apoptosis induced by a pro-inflammatory cytokine mix [IL- I beta (interleukin- I beta), TNF-alpha (tumour necrosis factor-a) and IFN-gamma (interferon-gamma)] was additionally assessed by flow cytometry. Culture for 24 h with 10 MM L-alanine resulted in desensitization to the subsequent acute insulin stimulatory effects Of L-alanine. This was accompanied by substantial changes in gene expression of BRIN-BD I I cells. Sixty-six genes were up-regulated; 1.8-fold, including many involved in cellular signalling, metabolism, gene regulation, protein synthesis, apoptosis and the cellular stress response. Subsequent functional experiments confirmed that L-alanine provided protection of BRIN-BD I I cells from pro-inflammatory cytokine-induced apoptosis. Protection from apoptosis was mimicked by NMA or DPI suggesting L-alanine enhances intracellular antioxidant generation. These observations indicate important long-term effects of L-alanine in regulating gene expression, secretory function and the integrity of insulin-secreting cells. Specific amino acids may therefore play a key role in beta-cell function in vivo.