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

H. J L Frank, W. M. Pardridge, W. L. Morris, Ronald (Ron) Rosenfeld, T. B. Choi

Research output: Contribution to journalArticle

122 Citations (Scopus)

Abstract

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, and 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 105 ng/ml (17.8 μM). 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 M(r) that was abolished by 0.6 μg/ml (80 nM) unlabeled IGF I but not by 10.0 μg/ml (1780 nM) unlabeled insulin. Peptide internalization was monitored by the acid-wash 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. These studies show that brain capillaries have separate receptors for insulin, IGF I, and IGF II. 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.

Original languageEnglish (US)
Pages (from-to)654-661
Number of pages8
JournalDiabetes
Volume35
Issue number6
StatePublished - 1986
Externally publishedYes

Fingerprint

Somatomedins
Microvessels
Insulin-Like Growth Factor II
Insulin-Like Growth Factor I
Insulin
Brain
IGF Type 1 Receptor
Peptides
High Pressure Liquid Chromatography
IGF Type 2 Receptor
Trichloroacetic Acid
Acids
Peptide Hormones
Insulin Receptor
Endocytosis
Blood-Brain Barrier
Radioactivity
Cerebrospinal Fluid
Polyacrylamide Gel Electrophoresis
Adenosine Triphosphate

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Frank, H. J. L., Pardridge, W. M., Morris, W. L., Rosenfeld, R. R., & Choi, T. B. (1986). Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels. Diabetes, 35(6), 654-661.

Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels. / Frank, H. J L; Pardridge, W. M.; Morris, W. L.; Rosenfeld, Ronald (Ron); Choi, T. B.

In: Diabetes, Vol. 35, No. 6, 1986, p. 654-661.

Research output: Contribution to journalArticle

Frank, HJL, Pardridge, WM, Morris, WL, Rosenfeld, RR & Choi, TB 1986, 'Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels', Diabetes, vol. 35, no. 6, pp. 654-661.
Frank HJL, Pardridge WM, Morris WL, Rosenfeld RR, Choi TB. Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels. Diabetes. 1986;35(6):654-661.
Frank, H. J L ; Pardridge, W. M. ; Morris, W. L. ; Rosenfeld, Ronald (Ron) ; Choi, T. B. / Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels. In: Diabetes. 1986 ; Vol. 35, No. 6. pp. 654-661.
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abstract = "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, and 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 105 ng/ml (17.8 μM). 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 M(r) that was abolished by 0.6 μg/ml (80 nM) unlabeled IGF I but not by 10.0 μg/ml (1780 nM) unlabeled insulin. Peptide internalization was monitored by the acid-wash 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. These studies show that brain capillaries have separate receptors for insulin, IGF I, and IGF II. 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.",
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