Internalization and release of insulin from hepatocytes.
Degradation of internalized insulin was studied after binding at 25 degrees C and 37 degrees C to isolated hepatocytes. The cells were washed to avoid extracellular insulin contamination. Degradation of both, intracellular and extracellular 125I-insulin, was measured with TCA and insulin antibody. In these conditions binding at 25 degrees C and 37 degrees C was equal but both intra and extracellular degradation were greater at 37 degrees C than at 25 degrees C. At both temperatures, intracellular degradation was greater than extracellular degradation with accumulation of degraded and non-degraded intracellular insulin. To study in what state hepatocytes release internalized insulin into the medium, 125I-insulin association was performed at an intermediate temperature (30 degrees C). Extracellular insulin contamination (whether associated or not) was avoided by three methods: 1) washing; 2) treatment with insulin degrading enzyme(s) and washing; 3) treatment with insulin degrading enzyme(s) then with trypsin and washing. Kinetics of radioactivity released from the cells was identical in the three conditions and the radioactivity was released throughout the experiments. Complete degradation of the released insulin was observed by gel filtration when the previous binding was 0.4 ng insulin/10(6) cells. When the dose of associated insulin increased (25 ng/10(6) cells) 3.5% of non-degraded insulin was liberated and when the dose was 14,300 ng/10(6) cells, the insulin released was 44.3%. In one experiment during the first 30 min, the insulin released was 52.88% and in the last 45 min 39.59%. To study the biologic behavior of the insulin released from cells, a group of mice were injected with this insulin (8.4 mU/mouse) and blood glucose was measured. The released insulin behaved as intact insulin as far as blood glucose responses were concerned. We may conclude that liver cells have the ability to internalize insulin and release biologically active insulin after accumulation.
Cresto
J C
JC
Unidad de Endocrinología y Fundación FLIP Hospital General de NiñosDr. Pedro Elizalde, Buenos Aires, Argentina.
Camberos
M C
MC
D'Alessandro
V
V
Basabe
J C
JC
D000818:Animals; D001786:Blood Glucose / Q000032:analysis; D002478:Cells, Cultured; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism; D013696:Temperature; D013748:Tetracaine / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D001786:Blood Glucose; D007328:Insulin; D013748:Tetracaine; D011972:Receptor, Insulin; D014357:Trypsin