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 [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.
Vu
L
L
Department of Medical Biochemistry, Department of Clinical Biochemistry, University Medical Centre, 1 rue Michel-Servet, Geneva 4, 1211, Switzerland.
Pralong
W F
WF
Cerini
F
F
Gjinovci
A
A
Stöcklin
R
R
Rose
K
K
Offord
R E
RE
Kippen
A D
AD
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000096:biosynthesis*; D007328:Insulin / Q000032:analysis*; D007339:Insulysin / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013997:Time Factors; D014316:Tritium / Q000378:metabolism
D007328:Insulin; D010446:Peptide Fragments; D014316:Tritium; D006003:Glycogen; D007339:Insulysin; D005947:Glucose