John N E, Andersen H U, Fey S J, Larsen P M, Roepstorff P, Larsen M R, Pociot F, Karlsen A E, Nerup J, Green I C, Mandrup-Poulsen T
School of Biological Sciences, University of Sussex, UK.
Diabetes. 2000 Nov;49(11):1819-29. doi: 10.2337/diabetes.49.11.1819.
Interleukin-1beta (IL-1beta) treatment of neonatal rat islets for 24 h induces changes in the expression of 105 of 2,200 proteins, as determined previously by two-dimensional (2D) gel electrophoresis. Nitric oxide (NO) has been implicated as one of the mediators of IL-1beta effects in insulin-containing cell lines and rat islets. The aims of this study were 1) to determine the involvement of NO in IL-1beta-induced alterations in protein expression and 2) to investigate the effects of chemically generated NO on protein expression by 2D gel electrophoresis of neonatal rat islet samples. IL-1beta-induced NO production was prevented by incubation of islets in arginine-free medium supplemented with the arginine analog NG-nitro-L-arginine. [35S]methionine-labeled islet proteins were separated using 2D gel electrophoresis and analyzed using the BioImage computer program. Analysis revealed that the expression levels of 23 protein spots of the 105 protein spots, altered by prior treatment with IL-1beta (60 U/ml) alone, were significantly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production was prevented. The effects of chemically generated NO were investigated by exposing islets to the NO donor GSNO (100 micromol/l) for 24 h before labeling with [35S]methionine and 2D gel electrophoresis. Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectable proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expression of up to 42 proteins is altered by cytokine-induced or chemically generated NO in the precise experimental conditions chosen and 2) that the majority of proteins altered by prior treatment with IL-1beta may be the result of NO-independent IL-1beta-mediated regulation of gene expression. This study demonstrates that the combination of 2D gel electrophoresis and mass spectrometry is a powerful tool in the identification of beta-cell proteins involved in the response to toxic mediators.
如先前通过二维（2D）凝胶电泳所确定的，用白细胞介素-1β（IL-1β）处理新生大鼠胰岛24小时可诱导2200种蛋白质中的105种蛋白质的表达发生变化。一氧化氮（NO）被认为是含胰岛素细胞系和大鼠胰岛中IL-1β作用的介质之一。本研究的目的是：1）确定NO是否参与IL-1β诱导的蛋白质表达改变；2）通过新生大鼠胰岛样品的2D凝胶电泳研究化学合成的NO对蛋白质表达的影响。通过在补充有精氨酸类似物NG-硝基-L-精氨酸的无精氨酸培养基中孵育胰岛，可防止IL-1β诱导的NO产生。使用2D凝胶电泳分离[35S]甲硫氨酸标记的胰岛蛋白，并使用BioImage计算机程序进行分析。分析显示，在防止NO产生时，单独用IL-1β（60 U/ml）预先处理后改变的105个蛋白点中的23个蛋白点的表达水平受到显著影响（P < 0.01 [n = 4]和P < 0.05 [n = 19]）。在用[35S]甲硫氨酸标记和进行2D凝胶电泳之前，将胰岛暴露于NO供体GSNO（100 μmol/l）24小时，以研究化学合成NO的作用。基于计算机的分析确定，在GSNO处理的胰岛中，总共1600种可检测蛋白质中的19种蛋白质的表达发生了改变（P < 0.01）。我们得出以下结论：1）在所选的精确实验条件下，细胞因子诱导的或化学合成的NO可改变多达42种蛋白质的表达；2）先前用IL-1β处理而改变的大多数蛋白质可能是由不依赖NO的IL-1β介导的基因表达调控所致。本研究表明，2D凝胶电泳和质谱联用是鉴定参与对毒性介质反应的β细胞蛋白的有力工具。
