Insulin-responsive cultured foetal-rat hepatocytes. Their preparation and characterization.
An improved non-perfusion method for the preparation of cultured foetal-rat hepatocytes is described. Digestion of the liver with collagenase and deoxyribonuclease I gave yields of 40 X 10(6) hepatocytes/g of liver. The plating efficiency of hepatocytes in medium with 10 microM-cortisol was 50%. Cell morphology and metabolism were maintained through 3 days of monolayer culture, with minimal contamination by haematopoietic cells or fibroblasts. The cultured cells bound and degraded 125I-insulin in a time- and dose-dependent manner. The estimated ED50 for competitive binding at 37 degrees C was 1.1 nM. Curvilinear Scatchard plots were observed, with estimates of 16 500 high-affinity sites (Kd = 813 pM) and 53 000 low-affinity sites (Kd = 23 nM) per cell. The cultured cells demonstrated a glycogenic response to insulin, with an estimated ED50 of 120 pM. The degree of glycogenic response to insulin varied with time in culture: 500% above basal on day 1, 200% on day 2, and only 150% on day 3. Cultured foetal cells also exhibited a time-dependent uptake of 2-aminoisobutyric acid, which, in contrast with previous reports with adult cells, was not stimulated by the presence of 10 nM-insulin. Cultured foetal hepatocytes may provide an interesting model with which to study the relationship between insulin-receptor binding and insulin action.
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D008112:Liver Glycogen / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D000596:Amino Acids; D007328:Insulin; D008112:Liver Glycogen; D005947:Glucose