Binding and Internalization of Insulin and Insulin-Like Growth Factors by Isolated Brain Microvessels

Isolated brain capillaries were used as a model system to test for binding and internalization of insulin and insulin-like growth factors (IGF) I and II. At 37°C, the maximum specific binding of the 125I-labeled peptides was 48.0 ± 0.8%/mg capillary protein for IGF I, 40.6 ± 1.4% for IGF II, and 15.1 ± 0.6% for insulin. The concentration of unlabeled peptide needed to cause a 50% decrease in the maximum binding (ID50)was 22 ng/ml (2.9 nM), 25 ng/ml (3.3 nM), and 7 ng/ml (1.2 nM) for IGF I, IGF II, andinsulin, respectively. Unlabeled insulin competed poorly for the IGF I receptor, requiring 5000 ng/ml (667 nM) to cause a 50% reduction in binding, and did not compete at all for the IGF II receptor at concentrations up to 105 ng/ml (17.8 μM). The IGF I receptor was further characterized by reduced polyacrylamide gel electrophoresis ofthe disuccinimidyl suberate cross-linked 125I-labeled IGF I receptor. The gel showed a distinct band at 133,000 M1 that was abolished by 0.6 μg/ml (80 nM) unlabeled IGF I but not by 10.0 ng/ml (1780 nM) unlabeled insulin. Peptide internalization was monitored by the acidwash technique. Only 22% of the bound IGF I was internalized, but 50% of the insulin and 43% of the IGF II were acid resistant. Capillaries prelabeled with internalized 125I-insulin could then export radioactivity into fresh, insulin-free media in a time- and temperature-dependent manner. However, high-performance liquid chromatography (HPLC) and trichloroacetic acid (TCA) analysis of the released material showedthat it consisted mostly of degraded peptide. These studies show that brain capillaries have separate receptors for insulin, IGF I, and IGFII. The endocytosis of these peptides is in the order of insulin > IGF II >IGF I, the same order as their distribution in cerebrospinal fluid (CSF) and brain, based on published data. Although the HPLC data do not support the general conclusion that the blood-brain barrier functions as a selective peptide-hormone transporter, this may be the result of cellular damage because the capillaries are ATP depleted.