L-Alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic beta-cell line and protects from pro-inflammatory cytokine-induced apoptosis.
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 cytokine-induced apoptosis were studied using clonal BRIN-BD11 cells. Expression profiling of BRIN-BD11 cells chronically exposed to L-alanine was performed using oligonucleotide microarray analysis. The effect of alanine, the iNOS (inducible nitric oxide synthase) inhibitor NMA (N(G)-methyl-L-arginine acetate) or the iNOS and NADPH oxidase inhibitor DPI (diphenylene iodonium) on apoptosis induced by a pro-inflammatory cytokine mix [IL-1beta (interleukin-1beta), TNF-alpha (tumour necrosis factor-alpha) 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-BD11 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-BD11 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.
Cunningham
Grainne A
GA
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
McClenaghan
Neville H
NH
Flatt
Peter R
PR
Newsholme
Philip
P
D000409:Alanine / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D016207:Cytokines / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009569:Nitric Oxide / Q000096:biosynthesis; D009573:Nitrites / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D026361:Reactive Nitrogen Species / Q000037:antagonists & inhibitors / Q000502:physiology; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
D016207:Cytokines; D007328:Insulin; D009573:Nitrites; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D000409:Alanine