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Endocrinology Vol. 147, No. 6 2611-2615
Copyright © 2006 by The Endocrine Society

Reciprocal Interactions of Insulin and Insulin-Like Growth Factor I in Receptor-Mediated Transport across the Blood-Brain Barrier

Yongmei Yu, Abba J. Kastin and Weihong Pan

Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana 70808

Address all correspondence and requests for reprints to: Weihong Pan, M.D., Ph.D., Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808. E-mail: weihong.pan{at}pbrc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the blood-brain barrier limits free passage of peptides and proteins from the peripheral circulation to the central nervous system, specific transport systems for insulin and IGF-I have been identified. To further determine whether insulin and IGF-I share the same transport system, and if not, whether the two transport systems interact with each other, we performed multiple-time regression analysis in mice after iv injection and in situ brain perfusion of these peptides. Insulin and IGF-I caused reciprocal inhibition of each other’s transport, although the effect of insulin was detected only by the in situ brain perfusion system. The interaction took place mainly at the step of cell surface binding as seen in cultured rat brain endothelium 4 brain microvessel endothelial cells. Further studies in 3T3 cells stably overexpressing the insulin receptor showed that the sharing of the transport systems was only partial. We conclude that insulin and IGF-I are mainly transported by their own transport systems, but a small amount can enter the brain by their "noncognate" transporters. The redundancy of their transport systems illustrates the regulatory function of the blood-brain barrier and reflects the importance of blood-borne insulin and IGF-I in the central nervous system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN IS MAINLY synthesized by ß-cells in the pancreas, whereas IGF-I is produced by liver and many other organs in the periphery (1, 2). Both insulin and IGF-I bind to insulin and IGF receptors that belong to a family of tyrosine kinase receptors. The affinity for their cognate receptors is 100-1000 times higher than that for other receptors in the family (3, 4). Signaling through the insulin receptor plays a major role in maintenance of metabolic homeostasis, while activation of the IGF-I receptor preferentially controls developmental and growth processes (5). In physiological situations, the blood concentrations of insulin and IGF-I are about 80 µU/ml (about 3 µg/ml) and 170 ng/ml, respectively (6, 7). Although IGF-I can be synthesized in the brain, little if any insulin is produced there (8, 9), so that it is likely that most central nervous system (CNS) effects exerted by these peptides result from their passage across the blood-brain barrier (BBB).

It was believed for many years that insulin did not cross the BBB. It took a series of experiments to establish that insulin does enter the brain from blood (10, 11, 12, 13). Evidence for the saturable nature of the BBB transport system for insulin was provided by a classic study in which its autoradiographic distribution in brain was inhibited by excess unlabeled insulin from blood (14). Specific binding of insulin to endothelial cells provided supplemental information suggesting that insulin receptors may be involved in the transport of insulin into the CNS (15). The pharmacokinetics of BBB permeation of insulin was quantified in several studies by Banks et al. (1, 16, 17, 18, 19). These studies showed that the blood-to-brain influx of insulin is saturable and can be modified by pathophysiological changes such as fasting and acute hyperglycemia.

The transport system for IGF-I has also been characterized (20, 21). Unlike the situation for insulin in which excess unlabeled insulin decreases the influx rate of 125I-insulin, excess unlabeled IGF-I enhances the influx rate of 125I-IGF-I. Such paradoxical effects are probably related to the presence of binding proteins in the peripheral circulation, as further shown by in situ brain perfusion studies (21). IGF receptors are also present at the BBB (20, 22). Signaling events downstream to insulin and IGF-I receptors in cerebral microvessel endothelial cells involve induction of endothelial nitric oxide synthase, production of vascular endothelial growth factor, and endothelin (23, 24). Because both insulin and IGF-I are present in blood, they might affect the transport of each other across the BBB, and such interactions at the BBB level could have important consequences for CNS function. Thus, we tested the hypothesis that insulin and IGF-I mainly cross the BBB by separate transport systems that can be modulated by each other.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adult male CD1 mice 5–6 wk of age were used following the protocol approved by the Institutional Animal Care and Use Committee. The mice were studied after anesthesia induced by ip injection of a mixture of ketamine, xylazine, and acepromazine. Insulin from bovine pancreas, BSA, and chloramine-T radiolabeling reagents were obtained from Sigma (St. Louis, MO). Recombinant mouse IGF-I was purchased from R&D Systems (Minneapolis, MN).

Radioactive labeling of compounds
Insulin and IGF-I were radioactively labeled with 125I by the chloramine-T method, with the reaction stopped at 1 min by addition of sodium metabisulfite, and purified on columns of Sephadex G-10 (25). The specific activity of 125I-insulin was about 100 Ci/g, and the specific activity of 125I-IGF-I was about 30 Ci/g. Albumin was radioactively labeled with 131I by the same method, with a specific activity of about 60 Ci/g. The labeled components were separated into aliquots and stored at –20 C until use.

Measurement of kinetics of transfer from blood to brain
Because IGF-I and insulin are peptides that have slow, flow-independent permeation across the BBB, we were able to determine the rate and extent of blood-to-brain transfer by multiple-time regression analysis (26, 27, 28). A group with only labeled peptide was studied with additional groups which contained excess unlabeled peptide in addition to the labeled peptide so as to identify potential modifiers of transport. These consisted of 2 µg/mouse of unlabeled IGF-I for the 125I-insulin study and 1 µg/mouse of unlabeled insulin for the 125I-IGF-I study. Because GH interacts with insulin and a variety of GH effects are mediated by IGF-1, an additional group with 10 µg/mouse of unlabeled GH was also included in the study of 125I-insulin or 125I-IGF-I. In each group of mice (n = 6–8 per group, representing different time points), a bolus of 100 µl lactated Ringer’s with 1% BSA containing 20,000 cpm/µl 125I-insulin or 125I-IGF-I was delivered into the left jugular vein at time 0. At various times between 1–20 min, blood was collected by dissection of the right carotid artery and the mouse was decapitated immediately afterward. The radioactivity in the whole brain and 50 µl of serum was measured, and the brain to serum ratio of 125I-insulin or 125I-IGF-I in each gram of brain was calculated separately. Based on the exponential decay pattern of serum radioactivity, the exposure time was calculated as the integral of serum radioactivity over time divided by the serum radioactivity at each experimental time. This represents the theoretical value correlated with each experimental time t if the blood concentration of 125I-peptide was constant from time 0 to time t. The linear regression correlation between the brain to serum ratio and exposure time was determined with Prism 3.03 (GraphPad Software, San Diego, CA). The unidirectional influx rate Ki, the slope of the linear regression line, reflects how fast the peptide permeates the BBB. The initial volume of distribution Vi, the y-intercept, illustrates the cerebral vascular volume of the particular peptide. Differences of the regression lines between groups were compared by the least square method with the Prism 3.03 program.

For in situ brain perfusion, the descending aorta was clamped and the bilateral jugular veins were severed. Two groups of mice were studied simultaneously: radiotracer only (125I-IGF-I plus 131I-albumin, 1 µCi/ml each) and radiotracer plus excess unlabeled insulin or IGF-I (2 µg/ml, or at doses specified in Results). The perfusion time was 5 min, and the perfusion rate was 2 ml/min, driven by a perfusion pump (KD Scientific, Holliston, MA; model 780100S). The perfusion buffer was preoxygenated except that hemoglobin was not included (29, 30). Each mouse received 2 min of prewash with buffer only and another minute of postwash to remove blood components and residual radiotracers from the cerebral vasculature, respectively. At the end of the procedure, the mouse was decapitated, and the brain to perfusate ratio of radioactivity per gram of brain was measured. Statistical analysis was performed as described in the preceding paragraph.

In vitro transport assays
125I-insulin and 125I-IGF-I binding and endocytosis assays were performed in rat brain endothelium 4 (RBE4) (kind gift from Dr. Pierre-Olivier Couraud, Institute Cochin and NeuroTech, Paris, France) and NIH 3T3 cells overexpressing the human insulin receptor (3T3-InsR; kind gift from Dr. Jianping Ye at Pennington Biomedical Research Center, Baton Rouge, LA). The group with only 125I-insulin or 125I-IGF-I was studied simultaneously (triplicate wells per group) with the following potential modulators: excess unlabeled IGF-I (500 ng/ml) or insulin (500 ng/ml). Equal numbers of RBE4 cells were seeded to six-well plates precoated with rat tail collagen type I. Radiotracer uptake assays were performed when the cells grew confluent. The culture medium for RBE4 cells contained equal amounts of {alpha}MEM and F10, supplemented with 1 ng/ml of basic fibroblast growth factor, antibiotic/antimycotic mix, 0.3 mg/ml of geneticin, and 10% FBS. The medium for 3T3-InsR cells was high-glucose DMEM containing antibiotics and 10% FBS. The cells were preequilibrated for 15 min in 1 ml of transport buffer, which was the basic medium supplemented with 25 mM HEPES and 0.5% BSA.

For binding assays, 125I-insulin or 125I-IGF-I (900,000 cpm/ml) were added in 1 ml of transport buffer and incubated with the cells at 4 C for 3 h. Afterward, the cells were washed with ice-cold PBS. Cell surface binding was determined by incubation of the cells in ice-cold stop-strip buffer (0.2 N acetic acid in PBS, pH 2.5) for 10 min. The radioactivity in the stop-strip buffer was expressed as a percentage of the total radioactivity added to the cells. The percent binding was normalized by the amount of protein in each well.

For endocytosis assays, the cells were equilibrated in transport buffer on ice for 20 min, and incubated with pulsing buffer containing 125I-insulin or 125I-IGF-I (900,000 cpm/ml) in transport buffer for 3 h at 4 C. The unbound radiotracers were then removed by aspiration and careful wash of the cells with ice-cold PBS twice. Prewarmed transport buffer was added, and the internalization of surface-bound radiotracer at 37 C was measured 20 min later by removal of the remaining surface binding with ice-cold stop-strip buffer (0.2 N acetic acid in PBS, pH 2.5) and lysis of cells to obtain the internalized fraction. The maximal potential internalization represents the sum of the acid-resistant binding and the amount internalized, and it is expressed as the percent total radioactivity added to the well. Each study group contained triplicate wells of cells. Group means are expressed with their SEs, and statistically significant changes were determined by an overall ANOVA for each fraction, followed by Tukey’s post hoc test.

Western blotting
Confluent 3T3-InsR and RBE4 cells grown on 10-cm dishes were washed with PBS and lysed with M-PER lysis buffer (Pierce, Rockford, IL). Protein (96 µg/well) was loaded onto 6% SDS-PAGE and electrophoresed. The proteins were transferred to nitrocellulose membrane and incubated with 2 µg/ml primary antibody against the C terminus of the ß-subunit of the human insulin receptor (Santa Cruz Biotechnology, Santa Cruz, CA). To quantify the relative level of protein expression, an antibody against ß-actin was also used. The signal was detected by use of HRP-conjugated secondary antibodies and the Western lightning chemiluminescent reagent (PerkinElmer Life Science, Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Influx rate of 125I-insulin from blood to brain was significantly decreased by IGF-I
The blood-to-brain influx of 125I-insulin was linear during the study period (1–20 min after iv injection in mice). As shown in Fig. 1Go, the influx rate was 0.80 ± 0.16 µl/g·min, and the initial volume of distribution was 14.2 µl/g. Of the total amount injected iv, the brain uptake was about 0.1% per gram of brain at 20 min, in the range of most peptides. Given the specific activity of 100 Ci/g of 125I-insulin and the injection amount of 0.91 µCi, about 9.1 pg of insulin was present in a gram of brain tissue. The results indicate that a significant amount of 125I-insulin reached the brain. Addition of 2 µg/mouse of unlabeled IGF-I (about 220-fold excess) to the 125I-insulin injection solution significantly decreased the rate of entry (Ki = 0.31 ± 0.12 µl/g·min, P < 0.05). This effect was specific, because unlabeled GH did not cause significant reduction of the influx rate of 125I-insulin in the same study (Ki = 0.69 ± 0.18 µl/g·min, P > 0.05).


Figure 1
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  		FIG. 1. Influx of 125I-insulin after iv bolus injection into mice. The influx rate in the group with only 125I-insulin was 0.80 ± 0.16 µl/g·min. Excess IGF-I at 220-fold excess caused a significant inhibition of this influx. Excess GH at 1100-fold excess did not cause significant changes.

 
To further determine the possible modulation of 125I-IGF-I influx by insulin, multiple-time regression analysis was performed in three groups of mice (n = 8 per group). The influx rate in the control group with only 125I-IGF-I was 0.53 ± 0.13 µl/g·min, and the initial volume of distribution was 19.3 µl/g. The coinjection of neither unlabeled insulin (an excess of 1 µg/mouse, in contrast to about 0.03 µg 125I-IGF-I) nor GH (10 µg/mouse) had any significant effect on the influx rate or initial volume of distribution of 125I-IGF-I after iv injection (Fig. 2Go).


Figure 2
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  		FIG. 2. Influx of 125I-IGF-I after iv bolus injection into mice. The influx rate (0.53 ± 0.13 µl/g·min) in the group with only 125I-IGF-I was not significantly changed in the presence of 110-fold excess insulin or 1100-fold excess GH.

 
Insulin decreased the uptake of 125I-IGF-I tested by in situ brain perfusion
To eliminate the possibility in the iv studies that serum IGF-I binding proteins or a potential hypoglycemic effect of insulin masked the potential inhibitory effect of insulin on the transport of 125I-IGF-I, in situ brain perfusion was performed with serum-free buffer. Two groups were studied (n = 5 per group): mice receiving 125I-IGF-I and 131I-albumin only and mice receiving 2 µg/ml of unlabeled insulin in addition. After 5 min of perfusion, excess insulin caused a significant decrease in the entry of 125I-IGF-I into the brain (P < 0.05) (Fig. 3Go).


Figure 3
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  		FIG. 3. Influx of 125I-IGF-I as determined by in situ brain perfusion and normalized by subtraction of 131I-albumin. The group receiving only 125I-IGF-I had a brain to perfusate ratio of 15.48 ± 1.30 µl/g, whereas the ratio for the group with additional insulin (2 µg/ml) was 10.11 ± 1.01 µl/g. The difference between the groups was significant (*, P < 0.01).

 
Reciprocal inhibition of cell surface binding of 125I-insulin and 125I-IGF-I in RBE4 cells
Cell surface binding of 125I-IGF-I (1.45 ± 0.05%) was higher (P < 0.001) than that of 125I-insulin (0.92 ± 0.04%) in RBE4 cells. There was reciprocal inhibition by an excess of each unlabeled peptide. The concentration of 125I-insulin in the transport buffer (700,000 cpm/ml, with specific activity of 100 Ci/g) was about 3.2 ng/ml. Therefore, 500 ng/ml of unlabeled IGF-I represented more than a 156-fold excess. Similarly, 125I-IGF-I had a specific activity of 30 Ci/g, and thus the concentration was about 10 ng/ml. Unlabeled insulin at 500 ng/ml represented about a 50-fold excess. In both treatment groups there was a small, yet significant inhibition of cell surface binding (P < 0.05) (Fig. 4Go).


Figure 4
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  		FIG. 4. Binding of 125I-insulin in RBE4 endothelial cells 3 h after incubation at 4 C was significantly reduced in the presence of 500 ng/ml unlabeled IGF-I (*, P < 0.05). Binding of 125I-IGF-I was significantly higher than that of 125I-insulin (***, P < 0.001) and could be significantly reduced by 500 ng/ml unlabeled insulin (**, P < 0.01).

 
Binding and endocytosis of 125I-insulin in 3T3-InsR cells
Although Western blotting showed that RBE4 cells express the insulin receptor, the level of expression is low compared with 3T3-InsR cells in which the human insulin receptor is stably overexpressed (Fig. 5Go). Thus, the 3T3-InsR cells were used to further determine the potential interactions between insulin and IGF-I during endocytosis.


Figure 5
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  		FIG. 5. Expression of insulin receptors in 3T3-InsR cells and RBE4 cells as shown by Western blotting.

 
Cell surface binding of 125I-insulin was measured by incubation of the cells with radiotracer for 3 h at 4 C. Excess unlabeled IGF-I (500 ng/ml) caused a significant decrease in 125I-insulin binding (P < 0.01), and the effect of excess unlabeled insulin (500 ng/ml) was even more pronounced with about a 12-fold decrease (P < 0.001). Cotreatment of IGF-I and insulin was more effective than IGF-I treatment alone, but did not further decrease the binding of 125I-insulin when compared with insulin treatment alone (Fig. 6AGo).


Figure 6
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  		FIG. 6. A, Binding of 125I-insulin in 3T3-InsR cells 3 h after incubation at 4 C was significantly decreased in the presence of excess unlabeled IGF-I (500 ng/ml), insulin (500 ng/ml), or both. **, P < 0.01; ***, P < 0.001. B, Binding and internalization of 125I-insulin in 3T3-InsR cells 3 h after incubation at 4 C followed by 20 min of endocytosis at 37 C, Excess unlabeled IGF-I (500 ng/ml), insulin (500 ng/ml), or both peptides each significantly inhibited binding and internalization. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
An endocytosis assay was performed after incubation of the 3T3-InsR cells with 125I-insulin at 4 C for 3 h. The surface-bound 125I-insulin was allowed to internalize for 20 min at 37 C. At the end of the study, cell surface binding of 125I-insulin was significantly lower in the IGF-I (P < 0.05), insulin (P < 0.001), and IGF-I plus insulin (P < 0.001) groups than in the control. This is reflected by decreased internalization at this time and reduced maximal potential internalization (Fig. 6BGo). The results show that insulin had a greater effect than IGF-I in displacing 125I-insulin from its binding sites and in competing with 125I-insulin for endocytosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study investigated the interactions of insulin and IGF-I in receptor-mediated transport across the BBB. In previous studies, we have quantified the passage of intact insulin and IGF-I across the mouse BBB by saturable transport systems (1, 16, 17, 18, 19, 21). Peptides, polypeptides, and proteins have a low degree of relatively slow permeation, so that extraction by the brain cannot be accurately measured by arterial-venous differences. With multiple-time regression analysis of brain uptake from arterial blood, and mathematical modeling of the exposure time, we are able to sensitively determine BBB permeation (26, 27, 31). In the current study, the influx rates of insulin (0.80 ± 0.16 µl/g·min) and IGF-I (0.53 ± 0.13 µl/g·min) were comparable to those previously reported. The higher influx rate of insulin suggests that the transport system for insulin is more efficient than that for IGF-I.

The influx rate of insulin was decreased significantly in the presence of unlabeled IGF-I. This suggests that IGF-I can compete with 125I-insulin for transport across the BBB. However, in the same experimental setting of multiple-time regression analysis after iv bolus injection, excess unlabeled insulin failed to inhibit the influx rate of 125I-IGF-I. This is consistent with our previous observation that excess IGF-I cannot decrease the influx of 125I-IGF-I after iv injection, probably because of the presence of serum binding proteins. Therefore, we further determined the potential effect of insulin on the transport of 125I-IGF-I by use of an in situ brain perfusion system free of serum and its binding proteins. In such a perfusion system, there was a significant decrease in the uptake of 125I-IGF-I in the presence of insulin. Thus, reciprocal inhibition of the transport of insulin and IGF-I can occur at the BBB.

Studies in both mice and RBE4 cells showed that the transport system for insulin has a higher capacity than that for IGF-I. The higher specific binding of the IGF-I receptor compared with the insulin receptor is in agreement with the results of others (4, 32). It has been shown that endothelial-specific single receptor knockouts for either insulin or IGF-I are viable, have an intact BBB blocking uptake of Evan’s blue dye, and show a normal distribution of tight junction proteins (23, 24). The partial overlap of the transport systems for insulin and IGF-I at the BBB are probably neuroprotective against the loss or excess of one of the peptides in the brain.

The receptors for insulin and IGF-I are involved in the transport of each other; have similar structures, and can activate many of the same signaling cascades. When insulin and IGF-I receptors are expressed in the same cells, it is difficult to separate the actions of insulin and IGF-I, because these ligands can bind to either receptor, although with a higher affinity to their cognate receptors than to the other. To isolate the individual contributions of the insulin and IGF-I receptors in the transport process, we further performed binding and internalization studies on 3T3-InsR cells which have a high level of surface expression of the insulin receptor as shown in Fig. 6AGo. The results show that excess unlabeled insulin was a much more potent inhibitor than IGF-I for the endocytosis of 125I-insulin. Therefore, the reciprocal inhibition of receptor-mediated transport of insulin and IGF-I is only partial. This means that a small percent of insulin can be transported by the separate transport system for IGF-I, and a small percent of IGF-I can be transported by the separate transport system for insulin. The redundancy and partial compensation of these two specific transport systems illustrate the importance of the BBB transport and CNS functions of these two peptides.

In summary, we have used adult mice, cultured cerebral microvessel endothelial cells, and cells stably overexpressing the insulin receptor to show that insulin and IGF-I cross the BBB by separate transport systems with partial overlap. The coexistence of the transport systems and their redundant functions indicate that the BBB is an important mediator linking circulating peptides to their CNS actions.


   Acknowledgments
 
The RBE4 cell line was a kind gift from Dr. Pierre-Olivier Couraud (Cochin, Paris, France). The 3T3-InsR cell line was generously provided by Dr. Jianping Ye in Pennington Biomedical Research Center. We thank Dr. Jinhua Yan for Western blotting of the insulin receptor.


   Footnotes
 
This work was supported by National Institutes of Health Grants (NS45751 and NS46528 to W.P., and DK54880 and AA12865 to A.J.K).

Y.Y., W.P., and A.J.K. have nothing to declare.

First Published Online February 23, 2006

Abbreviations: BBB, Blood-brain barrier; CNS, central nervous system; RBE4, rat brain endothelium 4.

Received January 5, 2006.

Accepted for publication February 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Physiol Rev, July 1, 2007; 87(3): 873 - 904.
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W. A. Banks
Denial versus dualism: the blood-brain barrier as an interface of the gut-brain axis.
Endocrinology, June 1, 2006; 147(6): 2609 - 2610.
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