Vu L, Pralong W F, Cerini F, Gjinovci A, Stöcklin R, Rose K, Offord R E, Kippen A D
Department of Medical Biochemistry, Department of Clinical Biochemistry, University Medical Centre, 1 rue Michel-Servet, Geneva 4, 1211, Switzerland.
Anal Biochem. 1998 Aug 15;262(1):17-22. doi: 10.1006/abio.1998.2731.
We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [3H]glycogen from d-[6-3H]glucose. The dose-response curve of insulin in this assay exhibited an EC50 of 0.42 (+/-0.04) nM, which is comparable to the dissociation constant of insulin from its receptor in hepatocytes. We used this assay to examine possible residual insulin-like activity of the four major fragments formed upon insulin degradation by insulin protease. Fragments A1-13B1-9, A1-14B1-9,and A14-21B14-30 showed no measurable activity. Although preparations of fragment A14-21B10-30 displayed dose-dependent agonist activity with an EC50 of 380 (+/-40) nM, we conclude that this was due to an insulin-like impurity since the chemically synthesized fragment showed no such activity. In summary, this bioassay demonstrates the action of insulin on glycogen formation in hepatocytes and provides a rapid and sensitive measurement of insulin-like activity which could facilitate screening studies.
我们描述了一种通过测定由d-[6-³H]葡萄糖生成的[³H]糖原，来测量大鼠肝细胞原代培养物中特定胰岛素样活性的新型生物测定法。该测定法中胰岛素的剂量-反应曲线显示，半数有效浓度（EC50）为0.42（±0.04）nM，这与胰岛素在肝细胞中与其受体的解离常数相当。我们使用该测定法来检测胰岛素蛋白酶降解胰岛素后形成的四个主要片段可能存在的残余胰岛素样活性。片段A1-13B1-9、A1-14B1-9和A14-21B14-30未显示出可测量的活性。尽管片段A14-21B10-30的制剂表现出剂量依赖性激动剂活性，EC50为380（±40）nM，但我们得出结论，这是由于一种胰岛素样杂质所致，因为化学合成的片段没有这种活性。总之，这种生物测定法证明了胰岛素对肝细胞中糖原形成的作用，并提供了一种快速且灵敏的胰岛素样活性测量方法，这可能有助于筛选研究。
