Diabetes, Vol 35, Issue 6 654-661, Copyright © 1986 by American Diabetes Association

ARTICLES

Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels

HJ Frank, WM Pardridge, WL Morris, RG Rosenfeld and TB Choi

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 degrees 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, and insulin, 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 10(5) ng/ml (17.8 microM). The IGF I receptor was further characterized by reduced polyacrylamide gel electrophoresis of the disuccinimidyl suberate cross-linked 125I-labeled IGF I receptor. The gel showed a distinct band at 133,000 Mr that was abolished by 0.6 microgram/ml (80 nM) unlabeled IGF I but not by 10.0 micrograms/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 showed that it consisted mostly of degraded peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
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