Regulation of insulin receptor internalization in vascular endothelial cells by insulin and phorbol ester.
Phorbol 12-myristate 13-acetate (PMA) was used to examine the role of insulin receptor phosphorylation in the regulation of insulin receptor internalization in vascular endothelial cells. Association of 125I-insulin in rat capillary and bovine aortic endothelial cells preincubated with PMA was increased by 80 and 64% over control, respectively. The increase was due to enhanced 125I-insulin internalization as opposed to an effect on surface-bound hormone. PMA had no significant effect on 125I-insulin degradation or on release of internalized insulin from the cells. Internalization of 125I-labeled insulin receptor was determined by the resistance of labeled receptor to trypsinization. At 10 degrees C, nearly all of the labeled receptor was sensitive to removal by trypsin, indicating that it was exposed on the cell surface. Exposure of labeled cells to insulin (100 nM) at 37 degrees C resulted in the rapid appearance of trypsin-resistant insulin receptor, indicating receptor internalization. Steady state for receptor internalization was attained at 10-15 min. When surfaced-labeled cells were preincubated with PMA at 37 degrees C, the rate of insulin receptor internalization was increased by 3.6 +/- 0.2-fold and 2.1 +/- 0.5-fold at 1 and 5 min of insulin exposure, respectively (ED50 at 16 nM PMA). This effect of PMA was associated with an increase in serine phosphorylation of the insulin receptor. Thus, PMA increased insulin internalization in the endothelial cells by modulating the insulin-induced internalization of the receptor. The additive effects of PMA and insulin on insulin receptor phosphorylation suggest that the phorbol ester and insulin act via independent signaling mechanisms.
D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D007328:Insulin; D014443:Tyrosine; D012694:Serine; D011972:Receptor, Insulin; D014357:Trypsin; D013755:Tetradecanoylphorbol Acetate