SHORT-TERM INSULIN-INDUCED GLYCOGEN FORMATION IN PRIMARY HEPATOCYTES AS A SCREENING BIOASSAY FOR INSULIN ACTION

We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [H-3]glyco gen from D-[6-H-3]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 insuli n protease. Fragments A(1-13)B(1-9), A(1-14)B(1-9), and A(14-21)B(14-3 0) showed no measurable activity. Although preparations of fragment A( 14-21)B(10-30) displayed dose-dependent agonist activity with an EC50 of 380 (+/-40) nM, we conclude that this was due to an insulin-like im purity since the chemically synthesized fragment showed no such activi ty. In summary, this bioassay demonstrates the action of insulin on gl ycogen formation in hepatocytes and provides a rapid and sensitive mea surement of insulin-like activity which could facilitate screening stu dies. (C) 1998 Academic Press.