Processing and Release of Insulin and Insulin-Like Growth Factor I by Macro- and Microvascular Endothelial Cells*

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Abstract
Insulin and insulin-like growth factor I (IGF-I) processing by macro- and microvascular endothelial cells was investigated. Specific binding of insulin and IGF-I on the cepillary endothelial cells derived from rat fat pads was 4 .+-. 0.5% (.+-. SE) and 4.3 .+-. 0.3%/mg protein, respectively, in contrast to bovine aortic endothelial cells, which bound 9.3 .+-. 0.3% IGF-I/mg protein. Both binding and processing of insulin and IGF-I were time and temperature dependent in macro- and microvascular endothelial cells. After 30 min at 37.degree. C, between 40-50% of the bound IGF-I and insulin were internalized in both capillary and aortic endothelial cells, whereas 20-25% insulin and 15-20% IGF-I internalization were observed at 15 C. Less than 20% internalization was observed for both insulin and IGF-I at 4 C. Cellular inhibitors of hormone processing, such as chloroquine and monensin, enhanced cell-associated insulin at 37 C on the bovine aortic endothelial cells from 4.7% to 10.4 .+-. 1% and 9.9 .+-. 2% mg protein, respectively, at 60 min. Similarly, chloroquine and monensin increased the amount of [125I]IGF-I associated with aortic endothelial cells from 4.3 .+-. 0.2% to 5.5 .+-. 0.3% and 6.2 .+-. 0.7%/mg protein, respectively. Chloroquine and monensin increased [125I]insulin associated with rat capillary endothelial cells from a control of 2.9 .+-. 0.1% to 4.0 .+-. 0.2% and 3.8% .+-. 0.37%, respectively. No effect of chloroquine and monensin was observed on [125I]IGF-I binding to rat capillary endothelial cells. Leupeptin, a lysosomal protease inhibitor, did not affect insulin or IGF-I binding in either cell type. The internalized insulin and IGF-I were both rapidly released, with 70-80% of both hormones being detected in the medium by 120 min. The released hormones were mostly intact (> 80-90%), as assessed by trichloroacetic acid precipitability, gel filtration, and immunoprecipitation. Both insulin and IGF-I induced corresponding down-regulation of their receptors, as shown by a 66 .+-. 7% decrease in insulin binding in the capillary endothelial cells and a 72 .+-. 1% and 58 .+-. 1% decrease in IGF-I binding in the aortic and capillary endothelial cells, respectively. Thus, macro- and microvascular endothelial cells bind and process insulin and IGF-I by degradative and nondegradative pathways. The predominance of the nondegradative pathway for the processing of insulin and IGF-I and the modulation of their receptors by physiological hormone concentrations suggested that endothelial cells may regulate the access of insulin and IGF-I to their target cells.

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