0021-972X/78/0047-0699$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 47, No. 3 Printed in U.S.A.
INSULIN RECEPTORS IN HUMAN ENDOTHELIAL CELLS: IDENTIFICATION AND CHARACTERIZATION1 ROBERT S. BAR, JOHN C. HOAK and MICHAEL L. PEACOCK Divisions of Endocrinology and Hematology, U. of Iowa and VA Hosps., Iowa City, IA 52242 ABSTRACT: Specific binding of 125 I-insulin was found in cultured human endotheiial cells obtained from human umbilical veins. The binding reaction was rapid and reversible, demonstrated receptor site-site interactions of the negatively cooperative type, and was dependent on the temperature, pH and duration of incubation. At 21°C, steady-state conditions of binding occurred in 90 minutes, the pH optimum was 7.8 and less than 10% of the labeled hormone was degraded. Binding of tracer amounts of 125 I-insulin was inhibited by concentrations of unlabeled insulin as low as 0.2 ng/ml and 50% inhibition was' obtained at 2-5 ng/ml of unlabeled insulin. Unlabeled porcine insulin, 1 2 5 porcine proinsulin and desoctapeptide insulin inhibited the binding of Iporcine insulin in direct proportion to their biological potencies, whereas unrelated peptide hormones were without effect. In addition, insulin binding to the cells was inhibited by antibodies against insulin receptors. We conclude that human endothelial cells possess specific receptors for insulin whose physio-chemical properties are similar to those of insulin receptors in other tissues. rinsed with phosphate buffered saline (0.01M) then filled with 0.2% collagenase (Worthington Corp., Freehold, N.J.). After 6 minutes, the cell suspension was drained into 10 ml of MM199 medium with 20% fetal calf serum and the vein rinsed with an additional 40-50 ml of MM-199. The cell suspension was centrifuged, resuspended in MM-199 with 20% fetal calf serum and seeded. Cultures were incubated at 37°C in a 5% CO2 atmosphere. The medium was changed 24 hours later and the cultures inspected daily. Monolayers prepared by this technique have been shown to be greater than 90% endothelial cells, contain rare leukocytes and no fibroblasts (2). We and others have verified the specific endothelial nature of such cultures by morphological (light and electron microscopy, including the demonstration of WeibelPalade bodies), immunological (endothelialassociated antigens including factor VIII antigen) and biochemical criteria (production of prostacyclin) (2). 125 I-insulin Binding: Confluent primary cultures of endothelial cells were used for insulin binding studies
Endothelial cells line the intima of the vascular system and thus control, in part, the transport of macromolecules and nutrients between the blood and the subendothelial tissues. Because of the critical location of endothelial cells, abnormalities in their function have been postulated to be intimately involved in the pathogenesis of the vascular complications of diabetes niellitus. However, there are few direct studies to support such hypotheses. In the present report, we have utilized purified preparations of human endothelial cells to identify and characterize specific receptors for insulin. These findings suggest a potential role for insulin in the function of the endothelial cell. MATERIALS AND METHODS Endothelial Cell Preparation: Endothelial cells were prepared from the veins of human umbilical cords by a modification of the method of Jaffe et aj. (1). The umbilical vein was 'Submitted nay lu, ly/b.
699
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 31 August 2015. at 08:35 For personal use only. No other uses without permission. . All rights reserved.
JCE&M • 1978 Vol 47 • No 3
RAPID COMMUNICATIONS
700
3 days after seeding. Immediately prior to use in the binding assay, the cells were scraped from the flasks and resuspended in HEPES buffer (100 mM). This scraping procedure resulted in quantitative removal of adherent cells with subsequent cell viability of 85-90%. 125 I-insulin (140-220 yCi/yg insulin) was prepared by the chloramine T method (3). In competition studies, tracer amounts of 125I-insulin (0.2 ng/ml) were incubated with cells and varying concentrations of unlabeled insulin, in HEPES buffer (lOOmM). After 90 minutes of incubation at 21°C, pH 7.8, the cell-associated radioactivity was separated from the free hormone by centrifugation in a Beckman microfuge. "Nonspecific" binding, defined as the amount of 125 I-insulin bound in the presence of 10 5 ng/ml unlabeled insulin, was subtracted from total binding to yield specific binding. Proinsulin and desoctapeptide insulin (DOP) were kind gifts of Drs. R. Chance and F. Carpenter. RESULTS The binding of 125I-insulin to the endothelial cells was time and temperature dependent. When endothelial cells were incubated with 125I-insulin at 21°C, specific binding reach-
Figure 1: 125I-insulin binding to human endothelial cells at 21°C.
40
10 10"'
10'
10* IOJ [INSULIN]
IO4
(moles/L) 10s (ng/ml)
Figure 2: Competition data for 1 2 5 I . insulin binding to endothelial cells. Four separate studies are shown. ed a steady state by 60-90' and maintained at a constant level for 4 hours (Figure 1). The amount of 125 I-insulin bound at steady state was linearly related to cell number over a range of 0.4-4 x 10 6 cells/ml. For further studies of competition and specificity, 2 x 10"6eel Is/ml were incubated at 21°C for 90 minutes at pH 7.8. These conditions yielded steady-state binding, substantial specific binding of insulin, less than 10% degradation of labeled hormone (by precipitation with 5% trichloroacetic acid) and no change in pH or cell viability. In competition studies, maximal specific binding was =3% per 10 6 cells, with 50% inhibition of maximal binding caused by
Vol. 47, No. 3 Printed in U.S.A.
INSULIN RECEPTORS IN HUMAN ENDOTHELIAL CELLS: IDENTIFICATION AND CHARACTERIZATION1 ROBERT S. BAR, JOHN C. HOAK and MICHAEL L. PEACOCK Divisions of Endocrinology and Hematology, U. of Iowa and VA Hosps., Iowa City, IA 52242 ABSTRACT: Specific binding of 125 I-insulin was found in cultured human endotheiial cells obtained from human umbilical veins. The binding reaction was rapid and reversible, demonstrated receptor site-site interactions of the negatively cooperative type, and was dependent on the temperature, pH and duration of incubation. At 21°C, steady-state conditions of binding occurred in 90 minutes, the pH optimum was 7.8 and less than 10% of the labeled hormone was degraded. Binding of tracer amounts of 125 I-insulin was inhibited by concentrations of unlabeled insulin as low as 0.2 ng/ml and 50% inhibition was' obtained at 2-5 ng/ml of unlabeled insulin. Unlabeled porcine insulin, 1 2 5 porcine proinsulin and desoctapeptide insulin inhibited the binding of Iporcine insulin in direct proportion to their biological potencies, whereas unrelated peptide hormones were without effect. In addition, insulin binding to the cells was inhibited by antibodies against insulin receptors. We conclude that human endothelial cells possess specific receptors for insulin whose physio-chemical properties are similar to those of insulin receptors in other tissues. rinsed with phosphate buffered saline (0.01M) then filled with 0.2% collagenase (Worthington Corp., Freehold, N.J.). After 6 minutes, the cell suspension was drained into 10 ml of MM199 medium with 20% fetal calf serum and the vein rinsed with an additional 40-50 ml of MM-199. The cell suspension was centrifuged, resuspended in MM-199 with 20% fetal calf serum and seeded. Cultures were incubated at 37°C in a 5% CO2 atmosphere. The medium was changed 24 hours later and the cultures inspected daily. Monolayers prepared by this technique have been shown to be greater than 90% endothelial cells, contain rare leukocytes and no fibroblasts (2). We and others have verified the specific endothelial nature of such cultures by morphological (light and electron microscopy, including the demonstration of WeibelPalade bodies), immunological (endothelialassociated antigens including factor VIII antigen) and biochemical criteria (production of prostacyclin) (2). 125 I-insulin Binding: Confluent primary cultures of endothelial cells were used for insulin binding studies
Endothelial cells line the intima of the vascular system and thus control, in part, the transport of macromolecules and nutrients between the blood and the subendothelial tissues. Because of the critical location of endothelial cells, abnormalities in their function have been postulated to be intimately involved in the pathogenesis of the vascular complications of diabetes niellitus. However, there are few direct studies to support such hypotheses. In the present report, we have utilized purified preparations of human endothelial cells to identify and characterize specific receptors for insulin. These findings suggest a potential role for insulin in the function of the endothelial cell. MATERIALS AND METHODS Endothelial Cell Preparation: Endothelial cells were prepared from the veins of human umbilical cords by a modification of the method of Jaffe et aj. (1). The umbilical vein was 'Submitted nay lu, ly/b.
699
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 31 August 2015. at 08:35 For personal use only. No other uses without permission. . All rights reserved.
JCE&M • 1978 Vol 47 • No 3
RAPID COMMUNICATIONS
700
3 days after seeding. Immediately prior to use in the binding assay, the cells were scraped from the flasks and resuspended in HEPES buffer (100 mM). This scraping procedure resulted in quantitative removal of adherent cells with subsequent cell viability of 85-90%. 125 I-insulin (140-220 yCi/yg insulin) was prepared by the chloramine T method (3). In competition studies, tracer amounts of 125I-insulin (0.2 ng/ml) were incubated with cells and varying concentrations of unlabeled insulin, in HEPES buffer (lOOmM). After 90 minutes of incubation at 21°C, pH 7.8, the cell-associated radioactivity was separated from the free hormone by centrifugation in a Beckman microfuge. "Nonspecific" binding, defined as the amount of 125 I-insulin bound in the presence of 10 5 ng/ml unlabeled insulin, was subtracted from total binding to yield specific binding. Proinsulin and desoctapeptide insulin (DOP) were kind gifts of Drs. R. Chance and F. Carpenter. RESULTS The binding of 125I-insulin to the endothelial cells was time and temperature dependent. When endothelial cells were incubated with 125I-insulin at 21°C, specific binding reach-
Figure 1: 125I-insulin binding to human endothelial cells at 21°C.
40
10 10"'
10'
10* IOJ [INSULIN]
IO4
(moles/L) 10s (ng/ml)
Figure 2: Competition data for 1 2 5 I . insulin binding to endothelial cells. Four separate studies are shown. ed a steady state by 60-90' and maintained at a constant level for 4 hours (Figure 1). The amount of 125 I-insulin bound at steady state was linearly related to cell number over a range of 0.4-4 x 10 6 cells/ml. For further studies of competition and specificity, 2 x 10"6eel Is/ml were incubated at 21°C for 90 minutes at pH 7.8. These conditions yielded steady-state binding, substantial specific binding of insulin, less than 10% degradation of labeled hormone (by precipitation with 5% trichloroacetic acid) and no change in pH or cell viability. In competition studies, maximal specific binding was =3% per 10 6 cells, with 50% inhibition of maximal binding caused by
