Collins H, Najafi H, Buettger C, Rombeau J, Settle R G, Matschinsky F M
Diabetes Research Center, University of Pennsylvania School of Medicine, Philadelphia 19104-6015.
J Biol Chem. 1992 Jan 15;267(2):1357-66.
High resolution 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with computerized analysis of gel images was used to search for proteins whose biosynthesis was induced or repressed in pancreatic islet cells chronically exposed to high glucose in an in situ and a tissue culture model of islet cell adaptation to excessive fuel load. The in situ model involved a 4-day intravenous infusion of either 50% glucose or 0.45% saline solution, followed by islet isolation, [35S]methionine labeling at 3 and 18 mM glucose for both groups, and protein analysis by 2-dimensional SDS-PAGE. The tissue culture model involved a 7-day culture of isolated rat islets in RPMI 1640 with 10% fetal calf serum containing either 3 or 30 mM glucose, followed by radiolabeling and 2-dimensional PAGE of proteins as in the in situ model. A small fraction of about 1.5% of the approximately 2000 identifiable proteins can be characterized as adaptive proteins. Of these altogether 58 proteins in the two models, 5 proteins were demonstrable in both models and two of these (proteins 1526 and 7622) are particularly noteworthy. Protein 1526 (Mr 57,000; pI 5.09) showed the same response pattern in both models and its expression was most enhanced when islets from chronically glucose-infused animals or those cultured for 7 days at 30 mM were radiolabeled at 18 mM glucose. Protein 7622 (Mr 68,000; pI 6.50) (also known as GSP-65; Collins, H.W., Buettger, C., and Matschinsky, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5494-5498) showed a different labeling pattern in the two models: stimulation of [35S]methionine incorporation by 18 mM glucose both in control and experimental islets from the infusion study, but lack of such stimulation of radiolabeling in islets cultured for 7 days at 30 mM glucose in contrast to islets cultured at 3 mM. The experimental strategy and the methodology are evaluated and the significance of the results is discussed. Potentials of the approach and plans for future experiments are considered.
采用高分辨率二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）并结合凝胶图像的计算机分析，以寻找在胰岛细胞原位及组织培养模型中，长期暴露于高糖环境下其生物合成被诱导或抑制的蛋白质。胰岛细胞原位模型包括静脉输注50%葡萄糖或0.45%盐溶液4天，随后分离胰岛，两组均在3 mM和18 mM葡萄糖条件下进行[35S]甲硫氨酸标记，并通过二维SDS-PAGE进行蛋白质分析。组织培养模型是将分离的大鼠胰岛在含10%胎牛血清的RPMI 1640培养基中培养7天，培养基中葡萄糖浓度分别为3 mM或30 mM，随后进行放射性标记及二维聚丙烯酰胺凝胶电泳，方法同原位模型。在大约2000种可识别的蛋白质中，约1.5%的一小部分可被鉴定为适应性蛋白。在这两个模型中共鉴定出58种此类蛋白质，其中5种在两个模型中均能检测到，其中两种（蛋白质1526和7622）尤为值得关注。蛋白质1526（分子量57,000；等电点5.09）在两个模型中表现出相同的反应模式，当用18 mM葡萄糖对长期输注葡萄糖动物的胰岛或在30 mM条件下培养7天的胰岛进行放射性标记时，其表达增强最为明显。蛋白质7622（分子量68,000；等电点6.50）（也称为GSP-65；Collins, H.W., Buettger, C., and Matschinsky, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5494 - 5498）在两个模型中呈现出不同的标记模式：在输注研究的对照和实验胰岛中，18 mM葡萄糖均刺激[35S]甲硫氨酸掺入，但在30 mM葡萄糖条件下培养7天的胰岛中，与在3 mM条件下培养的胰岛相比，缺乏这种对放射性标记的刺激作用。对实验策略和方法进行了评估，并讨论了结果的意义。还考虑了该方法的潜力以及未来实验计划。
