Insulin binding in cultured Chinese hamster kidney epithelial cells. The effects of glucose concentration in the medium and tunicamycin.
An epithelial cell line established from a Chinese hamster kidney, CHK-ACE, was separated into two sublines, CHK-ACE-100 and CHK-ACE-400, by 18 successive passages in medium containing 100 and 400 mg/dl glucose, respectively. Binding of CHK-ACE-100 and CHK-ACE-400 cell to 125I-labeled insulin showed similar pH and time dependency; 125I-labeled insulin, concentration differed in the two sublines, however. Degradation of 125I-labeled insulin, as determined by its ability to bind insulin antibody and cells, was more extensive when preincubated with CHK-ACE-400 cell than with CHK-ACE-100 cells. When CHK-ACE-100 cells were grown in 400 mg/dl glucose for six passages, these cells showed more insulin binding sites than cells grown parallel in 100 mg/dl glucose; whereas CHK-ACE-400 cells grown in 100 mg/dl glucose for six passages showed fewer insulin binding sites than those grown parallel in 400 mg/dl glucose. A slight increase in Kf/Ke ratio was observed in both sublines when grown in 400 mg/dl glucose as compared to 100 mg/dl glucose, indicating attenuated negative cooperativity of the binding sites in cells grown in 400 mg/dl glucose. Tunicamycin, at concentrations from 0.016 to 0.125 micrograms/ml, showed no direct effect on the assay of 125I-labeled insulin binding to CHK-ACE-100 cells; exposure of CHK-ACE-100 cells to tunicamycin, at concentrations from 0.01 to 0.2 micrograms/ml, for 24 h caused a dose-dependent decrease in insulin binding capacity and an increase in Kf/Ke ratio. These data indicate that the number of insulin binding sites in the cultured Chinese hamster kidney epithelial cells increased with high glucose concentrations in the culture medium, whereas tunicamycin, an inhibitor of protein glycosylation, lowered the number of insulin binding sites.
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives*; D005947:Glucose / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology*
D003470:Culture Media; D007328:Insulin; D014415:Tunicamycin; D005947:Glucose; D005944:Glucosamine