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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-1389
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6561-6568
Copyright © 2005 by The Endocrine Society

Recombinant, Nonglycosylated Human Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3) Is Degraded Preferentially after Administration to Type II Diabetics, Resulting in Increased Endogenous Glycosylated IGFBP-3

D. R. Clemmons, M. Sleevi and W. H. Busby, Jr.

Department of Medicine, University of North Carolina School of Medicine (D.R.C., W.H.B.), Chapel Hill, North Carolina 27599; and Insmed, Inc. (M.S.), Richmond, Virginia 23060

Address all correspondence and requests for reprints to: Dr. David R. Clemmons, 6111 Thurston-Bowles, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599. E-mail: endo{at}med.unc.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Administration of IGF-binding protein-3 (IGFBP-3) with IGF-I stabilizes IGF-I concentrations and prolongs its half-life. One determinant of IGFBP-3 stability is proteolysis. Normal subjects have minimal IGFBP-3 protease activity; however, with pregnancy, acute catabolic illness, or diabetes, IGFBP-3 protease activity is increased.

Objective: This study was conducted to determine the degree of proteolysis that occurs in glycosylated, endogenous serum IGFBP-3 and nonglycosylated IGFBP-3 after administration of an IGF-I/IGFBP-3 combination to patients with diabetes.

Design: Thirty-two patients received either 1 (n = 8) or 2 (n = 24) mg/kg·d IGF-I/IGFBP-3 by bolus sc injection (n = 16) or continuous sc infusion (n = 16).

Results: When nonglycosylated IGFBP-3 was given, the abundance of both glycosylated and nonglycosylated forms of IGFBP-3 in serum was increased. Incubation of nonglycosylated IGFBP-3 with diabetic serum in vitro resulted in more rapid degradation compared with glycosylated IGFBP-3. When the serum obtained from subjects who had received nonglycosylated IGFBP-3 was analyzed, significant differences in the stability of glycosylated and nonglycosylated IGFBP-3 were present. The addition of increasing concentrations of nonglycosylated IGFBP-3 to diabetic serum resulted in a dose-dependent increase in the abundance of endogenous, glycosylated IGFBP-3. Administration of IGF-I and nonglycosylated IGFBP-3 for 2 wk to 32 subjects increased glycosylated IGF-I/IGFBP-3 by 20–40%. The increases were the greatest in the groups that received IGFBP-3 by infusion (e.g. 31% and 40%).

Conclusions: After administration to diabetics, nonglycosylated IGFBP-3 is degraded more rapidly than glycosylated IGFBP-3. By acting as a preferential substrate for the IGFBP-3 protease, nonglycosylated IGFBP-3 protects endogenous, glycosylated IGFBP-3 from degradation, allowing total IGFBP-3 concentrations to increase.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
ADMINISTRATION OF IGF-binding protein-3 (IGFBP-3) with IGF-I has been shown to prolong the half-life of IGF-I and to enhance IGF-I’s anabolic actions (1, 2, 3). Infusion of IGF-I/IGFBP-3 into patients with burns was shown to increase the ability of IGF-I to stimulate protein synthesis, and infusion into elderly patients with hip fractures increased bone accretion (3, 4). Similarly, administration of the IGF-I/IGFBP-3 complex to patients with type 1 diabetes has been shown to enhance insulin sensitivity (5). Although administration of IGF-I alone enhances insulin sensitivity, the administration of IGFBP-3 with IGF-I is equally efficacious and is associated with a lower incidence of acute side effects that are known to occur when IGF-I is given alone, such as development of edema, arthalgias, headaches, and retinal edema (3, 4, 5). This suggests that administration of IGFBP-3 with IGF-I alters the time course of the rate of increase in free IGF-I and possibly the rate of delivery of free IGF-I to tissues. Therefore, the factors that regulate the stability of IGFBP-3 in serum may be important for understanding how administration of IGFBP-3 alters IGF-I bioavailability.

One of the most important variables that determines the stability of IGFBP-3 in serum is proteolytic degradation (6, 7, 8). A calcium-dependent, serine protease that is abundant in serum obtained from pregnant subjects has been shown to degrade IGFBP-3 (7, 8). Increased proteolytic activity has also been shown to be present in patients during postoperative states (9, 10), during severe catabolic illness (11, 12), and with new-onset (13) as well as stable diabetes (14). In serum obtained from normal subjects, IGFBP-3 is degraded very slowly, and presumably part of this difference between serum from catabolic and normal subjects is due to a decrease in the amount of an endogenous inhibitor(s) (15). Some of these conditions, such as postoperative states, are associated with increases in free IGF-I. Although some of the IGFBP-3 fragments that remain in the circulation can bind to IGF-I, their affinity is reduced; thus, the bioavailability of IGF-I to extravascular tissues is enhanced (16). The rate of proteolytic cleavage of IGFBP-3 in diabetes is partly dependent on the degree of diabetic control; that is, when insulin is administered to poorly controlled diabetics, the rate of degradation is reduced (13). Recently, we completed a study in which the IGF-I/IGFBP-3 combination was given to subjects with type 2 diabetes to determine its effects on insulin sensitivity (17). The combination of drugs was given by either sc bolus injection or continuous sc infusion. We measured changes in IGFBP-3 concentrations by an RIA that measures both the endogenous, glycosylated and the exogenously administered, nonglycosylated forms of IGFBP-3. Because the total IGFBP-3 concentration did not increase in proportion to the total IGF-I concentration, these studies were undertaken to analyze the fate of the nonglycosylated IGFBP-3 that was administered and to determine the effect of administering nonglycosylated IGFBP-3 on the abundance of the endogenous, glycosylated form of the protein.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study design

Fifty-four patients with type 2 diabetes of an average duration of 17.1 yr were recruited. The baseline characteristics in this population and the results of treatment have been reported previously (17). All subjects provided written informed consent for a protocol that had been approved by the local institutional review board. Sera from eight subjects (selected randomly) from each of four separate treatment groups (total of 32 subjects) were analyzed for this report. After entry into the study, the subjects were fasted overnight before the first day of drug administration and then placed on a constant diet, which they continued for the 2-wk study period. They remained hospitalized during that time. Insulin dosage was adjusted based on capillary blood glucoses to avoid hypoglycemia and to maintain glycemic control. The subjects were randomized (using random number selection) to one of four treatment groups. Group 1 received a continuous sc infusion of IGF-I/ IGFBP-3 (2 mg/kg·d). Group 2 received the same total dosage as a 6-h infusion every 24 h. Subjects in group 3 received an sc injection of 1 mg/kg every 12 h, and subjects in group 4 received a single daily injection of 1 mg/kg at 1900 h. Mealtimes were standardized and occurred at 0800, 1200, and 1800 h. No subject lost significant weight during the trial. Repetitive sampling for IGF-I, free IGF-I, and IGFBP-3 was conducted on d 0, 1, 7, 14, and 15. The values for glucose, insulin, total and free IGF-I, as well as total IGFBP-3 have been previously reported (17). Briefly, those results showed that all four groups had significant and comparable reductions in insulin dosage and in fasting glucose. Groups 2 and 3 had small, but significant, reductions in postprandial glucose values, and the greatest decrease occurred in group 2. To determine the amount of IGFBP-3 proteolytic activity in the serum, blood samples were collected fasting at 0800 h and postprandially at 0900 and 2000 h on d 1 and 14. They were immediately centrifuged, and the serum was stored at –80 C.

Measurement of proteolysis

For analysis of proteolytic activity, 2 µl serum was incubated in a total volume of 100 µl 0.05 M Tris, containing 2 mM calcium chloride (pH 7.5). The incubation times varied between 24 and 72 h. Each experiment was repeated three times. For some experiments, nonglycosylated IGFBP-3, expressed in Escherichia coli and purified as previously described (18), or human glycosylated IGFBP-3, purified as previously described from transfected Chinese hamster ovary cell-conditioned medium (19), was added using final concentrations ranging from 0.25–2.5 µg/ml. In some experiments, the IGFBP-3 was deglycosylated by incubating 10 µl serum with 2 U protease-free N-glycanase (8 U/mg; Sigma-Aldrich Corp., St. Louis, MO) for 2 h at 37 C using a buffer described previously (19). The products of the reaction were either frozen and stored at –80 C before electrophoretic separation, or an aliquot was removed for additional analysis. For immunoblotting between 2 and 10 µl of the incubation mixture from each proteolytic reaction was added to 40 µl Laemmli sample buffer, and the sample was heated to 65 C for 2 min. The proteins were then separated by SDS-PAGE (7.5% gel) and transferred to a polyvinylidene difluoride membrane (0.45 µm pore size; Millipore Corp., Bedford, MA). The membrane was probed with a 1:1000 dilution of antihuman IGFBP-3 antiserum that had been prepared as described previously (19). The bands were visualized by enhanced chemiluminescence (Supra Signal CL-8 substrate system, Pierce Chemical Co., Rockford, IL), then exposed to film (Kodak XAR, Eastman Kodak Co., Rochester, NY) or read directly using a chemiluminescence image analysis system (Syngene, Inc., Frederick, MD). Signal intensities were then quantified by either scanning densitometry using NIH Image 1.63 (Bethesda, MD) or the software supplied with the chemiluminescence detector. Molecular weight standards (Invitrogen Life Technologies, Inc., Gaithersburg, MD) were run in a parallel lane. To quantify the changes that occurred in IGFBP-3 proteolysis over time, scanning densitometry was performed with an IGFBP-3 standard run in parallel on the same gel to standardize all exposure times. All data are expressed as corrected units, that is, corrected for changes in the intensity of the internal IGFBP-3 standards that were run on each gel to correct for differences in exposure times, gel loading, and protein transfer to the membranes.

Statistics

For single comparisons within the same group of subjects, a paired Student’s t test was used to determine significance. For comparisons of treatment effects among the four treatment groups, differences were analyzed using two-way ANOVA, followed by a t test with the Bonferroni correction.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
To identify the different forms of IGFBP-3 that were present in the serum of these subjects before and during treatment, two pools of serum samples obtained from six patients at the start of treatment and 12 h after IGF-I/IGFBP-3 administration were analyzed by SDS-PAGE with immunoblotting. As shown in Fig. 1Go, administration of nonglycosylated IGFBP-3 resulted in an increase in the amount of intact glycosylated IGFBP-3 with a molecular mass (Mr) estimate of 38–54 kDa and a decrease in the intensity of the 30-kDa glycosylated IGFBP-3 fragment. Similarly, a narrow band (Mr estimate, 34 kDa) was detected in the serum obtained 12 h after IGFBP-3 administration. Western ligand blotting confirmed that there was increased IGF-I-binding activity in the region of the 38- to 52-kDa band, suggesting that this increase was due to intact IGFBP-3. This result suggested that the administration of nonglycosylated IGFBP-3 was retarding the degradation rate of glycosylated IGFBP-3 and thus resulted in an increase in endogenous, glycosylated IGFBP-3. To confirm that the IGFBP-3 bands (Mr, 38–52 kDa) that were detected were glycosylated forms of IGFBP-3, protease-free N-glycanase was incubated with sera obtained before the injection of nonglycosylated IGFBP-3. As shown in Fig. 2Go, N-glycanase treatment resulted in a shift in the 38- to 54-kDa band to a 35- to 42-kDa band and the 28- to 32-kDa band shifted to a band with an Mr estimate of 27 kDa. The controls showed that N-glycanase treatment of pure glycosylated IGFBP-3 resulted in a similar change in its Mr estimate and that it did not result in a change in the Mr estimate of pure nonglycosylated IGFBP-3.



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  		FIG. 1. Administration of nonglycosylated IGFBP-3 increases the amount of glycosylated IGFBP-3 detected in serum. A pool of sera from six patients before (lanes 2 and 4) and 12 h after they had received an injection of nonglycosylated IGFBP-3 (lanes 3 and 5) was analyzed, and the abundance of endogenous, glycosylated IGFBP-3 as well as that of nonglycosylated IGFBP-3 was determined. The serum pool was analyzed by immunoblotting (lanes 2 and 3) and Western ligand blotting (lanes 4 and 5) using [125I]IGF-I for detection. An IGFBP-3 standard is shown in lane 1.

 


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  		FIG. 2. Deglycosylation of serum IGFBP-3. To confirm that the major form of IGFBP-3 present in serum was glycosylated, diabetic serum (lane 6) was treated with N-glycanase for 3 h, then analyzed by SDS-PAGE with immunoblotting (lane 5). Pure glycosylated IGFBP-3 (lane 1) was incubated with an equal concentration of N-glycanase for 3 h (lane 2). Lane 4 shows that pure deglycosylated IGFBP-3 (lane 3) is not degraded after exposure to N-glycanase.

 
We next determined whether there was a difference in the susceptibility of the endogenous, glycosylated and exogenously administered, nonglycosylated forms of IGFBP-3 to proteolysis. Sera from two patients obtained either 1 or 12 h after injection of nonglycosylated IGFBP-3 were incubated for 24 h at 37 C, and the band intensities of the intact forms of glycosylated and nonglycosylated IGFBP-3 were analyzed. When serum samples obtained 12 h after the IGF-I/IGFBP-3 injection were analyzed, the intensity of the intact, nonglycosylated IGFBP-3 band decreased by 82 ± 13% (P < 0.01), whereas the intact, glycosylated IGFBP-3 decreased by 32 ± 8% (P < 0.05) (Fig. 3Go). When the samples obtained 1 h after injection were compared, the decreases were 79 ± 11% (P < 0.01) and 24 ± 5% (P < 0.05), respectively.



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  		FIG. 3. Degradation of endogenous, glycosylated and nonglycosylated IGFBP-3 in diabetic serum. To analyze the stability of glycosylated IGFBP-3 and nonglycosylated IGFBP-3, sera obtained from two patients who had received nonglycosylated IGFBP-3 injections were incubated for 24 h and then analyzed. Sera obtained at 12 h (lanes 1, 3, 5, and 7) or 1 h (lanes 2, 4, 6, and 8) after the last IGFBP-3 injection were compared after no incubation (lanes 1–4) or after 24-h incubation (lanes 5–8). Glycosylated IGFBP-3 was degraded more slowly. In contrast, nonglycosylated IGFBP-3 disappeared more rapidly.

 
To confirm that glycosylation of IGFBP-3 in serum accounted for this difference in the rate of degradation and that it was not due to some other property of recombinant, nonglycosylated IGFBP-3, the degradation of endogenous glycosylated IGFBP-3 before and after exposure to N-glycanase was compared with that of nonglycosylated IGFBP-3 using a pool of sera obtained from six patients (Fig. 4Go). The intact, endogenous, glycosylated IGFBP-3 band intensity was reduced by 21 ± 5% after 24 h. Although some degradation of endogenous IGFBP-3 in serum occurred during the 2-h exposure to N-glycanase (lane 4), there was an additional 62 ± 8% reduction in intact IGFBP-3 band intensity during the subsequent 22-h incubation. When the decrease in intact IGFBP-3 (38–52 kDa) in the sample not exposed to N-glycanase after 24 h was compared with the decrease in the 36-kDa band in the sample that had been exposed to N-glycanase, there was a significant difference (P < 0.02). Additionally, the intensity of the IGFBP-3 fragment band was increased (e.g. 52 ± 5%) in the N-glycanase-treated sample, whereas in control serum, the glycosylated IGFBP-3 fragment band increased 13 ± 4% (P < 0.01). When nonglycosylated IGFBP-3 was added to the same pool of diabetic serum, there was a decrease of 49 ± 9% in intact, nonglycosylated IGFBP-3 band intensity after 24 h. These results suggest that the protease cleaves nonglycosylated IGFBP-3 more rapidly than endogenous glycosylated IGFBP-3. To exclude the possibility that concomitant administration of IGF-I influenced the degradation rate, the experiment was repeated with in vitro addition of nonglycosylated IGFBP-3 and IGF-I. The addition of IGF-I in vitro had no effect on the rate of glycosylated or nonglycosylated IGFBP-3 degradation (data not shown).



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  		FIG. 4. Comparison of degradation of endogenous, glycosylated IGFBP-3 before and after N-glycanase treatment and nonglycosylated IGFBP-3 after exposure to serum from diabetic patients. Sera from a pool of six patients with diabetes were incubated with no addition (lanes 1–5) or added nonglycosylated IGFBP-3 (1 µg/ml; lanes 6–8) for 0 (lanes 1 and 6), 24 (lanes 2 and 7), or 48 (lanes 3 and 8) h. Endogenous, glycosylated IGFBP-3 in serum was exposed to N-glycanase for 2 h (lane 4), then incubated for an additional 22 h (lane 5). The results show that intact, nonglycosylated IGFBP-3 (lanes 6–8) decreased by 49%, whereas glycosylated IGFBP-3 (lanes 1–3) decreased by 21%. The intensity of the N-glycanase-exposed endogenous IGFBP-3 band representing the intact form decreased by 62%.

 
To verify that nonglycosylated IGFBP-3 could inhibit glycosylated IGFBP-3 degradation, increasing concentrations of nonglycosylated IGFBP-3 (between 264-2640 ng/tube) were added in vitro with diabetic serum and incubated for 48 h at 37 C. As shown in Fig. 5Go, when intact, glycosylated IGFBP-3 in the serum was analyzed, there was a major decrease in the band intensity (e.g. 64 ± 8%) and a 62 ± 15% increase in the intensity of the 30-kDa fragment band after 48 h. The addition of increasing concentrations of nonglycosylated IGFBP-3 resulted in progressive increases in the intensity of the glycosylated IGFBP-3 band, and there was a marked increase in the intensities of the IGFBP-3 fragment bands. The Mr estimates of several of the fragment bands were similar to those noted when nonglycosylated IGFBP-3 was used as a substrate. This strongly suggests that nonglycosylated IGFBP-3 is being degraded preferentially by the protease and that this results in inhibition of degradation of intact, glycosylated IGFBP-3. When analyzed by scanning densitometry, the highest concentration of nonglycosylated IGFBP-3 tested induced a 99 ± 8% inhibition in the amount of glycosylated IGFBP-3 that was degraded after 48 h. We next compared the stability of the purified, nonglycosylated and endogenous, glycosylated forms of IGFBP-3 in serum. As shown in Fig. 6Go, added glycosylated IGFBP-3 had a stability in serum comparable to that of endogenous IGFBP-3, whereas added nonglycosylated IGFBP-3 was extensively degraded.



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  		FIG. 5. In vitro addition of nonglycosylated IGFBP-3 inhibits the degradation of glycosylated IGFBP-3. Sera from a pool of six patients were incubated for 24 (lane 2) or 48 (lanes 3 5, 6, and 7) h. Nonglycosylated IGFBP-3 was added in increasing concentrations (lane 5, 264 ng; lane 6, 1056 ng; lane 7, 2643 ng) before the incubation. Scanning densitometry showed that the intact IGFBP-3 band had decreased by 64 ± 8% at 48 h, and the 28–32 kDa fragment had increased by 62 ± 15% (lanes 1 and 3). The addition of increasing concentrations of nonglycosylated IGFBP-3 resulted in 58 ± 9% (lane 5), 79 ± 11% (lane 6), and 99 ± 8% (lane 7) inhibition of glycosylated IGFBP-3 cleavage. The experiment was repeated three times, and the results represent the mean ± SD of the three determinations.

 


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  		FIG. 6. Comparison of degradation of exogenously added, glycosylated and nonglycosylated IGFBP-3 in sera from diabetic patients. Because nonglycosylated IGFBP-3 appeared to be more labile, the sera from a pool of six patients with diabetes were incubated with no addition (lanes 1 and 4), added nonglycosylated IGFBP-3 (1 µg/ml; lanes 2 and 5), or added glycosylated IGFBP-3 (1 µg/ml; lanes 3 and 6) for 0 (lanes 1–3) or 48 (lanes 4–6) h. Pure nonglycosylated IGFBP-3 (lane 7) and pure glycosylated IGFBP-3 (lane 8) standards are shown. The results show that the added nonglycosylated IGFBP-3 (lanes 2 and 5) decreased by 48 ± 7%, whereas the added glycosylated IGFBP-3 (lanes 3 and 6) decreased by 5 ± 3%.

 
To determine whether administration of nonglycosylated IGFBP-3 affected the stability of glycosylated IGFBP-3 in the serum of patients treated in this study and whether there were any differences among the treatment groups, the samples obtained 0 and 12 h after drug administration on d 1 and 14 were compared for the abundance of intact, glycosylated IGFBP-3, the 30-kDa fragment of glycosylated IGFBP-3, and the 34-kDa intact, nonglycosylated IGFBP-3. The results from eight representative patients (two from each treatment group) are shown in Figs. 7Go and 8Go. When the 12-h point on d 1 was compared with the time zero point, the results show that the administration of nonglycosylated IGFBP-3 resulted in an increase in glycosylated IGFBP-3 that was detectible after 12 h, and the band intensity of the 30-kDa fragment of glycosylated IGFBP-3 was decreased. When the d 14 samples were analyzed, the intensity of the intact, glycosylated IGFBP-3 band was increased compared with that on d 1 at 0 h, and the nonglycosylated IGFBP-3 band was increased in all eight subjects compared with the d 1, 0 h samples.



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  		FIG. 7. Analysis of glycosylated and nonglycosylated IGFBP-3 band intensity in serum after in vivo administration of nonglycosylated IGFBP-3. Four diabetic patients (shown in A and B, lanes 1–4 or 5–8) are depicted. Immunoblots of these sera at various time points, including d 1, 0 h (lanes 1 and 5); d 1, 12 h (lanes 2 and 6); d 14, 0 h (lanes 3 and 7); and d 14, 12 h (lanes 4 and 8), are shown.

 


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  		FIG. 8. The results of four additional patients who received nonglycosylated IGFBP-3 are shown. The results are presented as they were in Fig. 7Go.

 
The changes in IGFBP-3 band intensity were analyzed for all 32 subjects by scanning densitometry, and the results are shown in Tables 1Go and 2Go. For d 1, intact, glycosylated IGFBP-3 increased between 5–14% at the 12 h point. The smallest increase was seen in group 3, and the greatest increases were noted in groups 1 and 2. The intergroup comparisons among groups 1, 2, and 4 did not show statistically significant differences, but all three groups showed a significantly greater increase compared with group 3 (P < 0.05). When the amount of intact, nonglycosylated IGFBP-3 that was present on d 1 after 12 h was added to the endogenous IGFBP-3 values, the percent increases in total, intact IGFBP-3 varied between 15–38% (Table 2Go). When the increases for groups 1 and 2 were compared, there was no difference, but both were significantly greater than the increases for groups 3 and 4 (P < 0.05). When the decreases in the 30-kDa glycosylated IGFBP-3 fragment band were compared, the greatest decrease was noted in group 2, and the smallest decreases were present in groups 1 and 4. When the abundance of the nonglycosylated, intact IGFBP-3 band across the four treatment groups was analyzed, the results showed that groups 1 and 2 had the greatest increases, and these increases were significantly greater than those in groups 3 and 4 (P < 0.05). Therefore, administration of IGFBP-3 by either 6- or 24-h infusion results in the highest levels of intact, exogenously administered IGFBP-3 after 12 h. When the d 14, 0 h values were compared with d 1, 0 h values, the increases in the abundance of intact endogenous, glycosylated IGFBP-3 varied between 9–16%. The sum of intact, glycosylated and nonglycosylated IGFBP-3 was increased between 22–48%. The greatest increases were present in groups 1 and 2, which were significantly greater than those in groups 3 and 4 (P < 0.05). The decreases in the band intensities of the 30-kDa fragment were similar to the d 1 changes (e.g. 31–53%). When the d 14, 12 h values were compared with the d 1, 0 h values, intact, glycosylated IGFBP-3 was significantly increased in each group (20–40%), and the sum of the intact, glycosylated plus nonglycosylated was also significantly increased (39–73%). The greatest increases occurred in groups 1 and 2. The abundance of the 30-kDa fragment was further decreased in each group (39–62%). When the changes on d 14 at 12 h were compared with the d 14, 0 h values, there was an additional significant increase in intact, glycosylated IGFBP-3 in groups 1–3 (P < 0.05). A significant increase in nonglycosylated IGFBP-3 was noted for group 4 (P < 0.05). The degree of decrease in the 30-kDa fragment band was significantly changed in groups 1 and 4 (P < 0.05).


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  		TABLE 1. Changes in intact glycosylated and nonglycosylated IGFBP-3 and in the 30-kDa fragment

 

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  		TABLE 2. Changes in IGFBP-3

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our findings show that nonglycosylated IGFBP-3 is degraded more rapidly than glycosylated IGFBP-3 in serum from patients with diabetes. More importantly, they demonstrate that if a sufficient amount of nonglycosylated IGFBP-3 is administered this slows the rate of degradation of endogenous, glycosylated IGFBP-3 in vivo. Therefore, a substantial percentage of the increase in total IGFBP-3 that we measured in our previous study by RIA after the administration of nonglycosylated IGFBP-3 is due to increases in endogenous, glycosylated IGFBP-3. Although we used four different treatment regimens, all four groups showed this increase, but it was the greatest in the two groups that received the IGFBP-3 as an infusion. Similarly, when the sums of the increases in intact glycosylated and nonglycosylated IGFBP-3 were compared, the greatest increase was seen in groups 1 and 2. The decrease in the intensities of the 28- to 32-kDa fragment of glycosylated IGFBP-3 was similar among the treatment groups, suggesting that they all had significant inhibition of intact, glycosylated IGFBP-3 cleavage. These findings suggest that when intact, nonglycosylated IGFBP-3 is administered, it is preferentially degraded by the IGFBP-3 protease, resulting in an increase in intact, glycosylated IGFBP-3. This conclusion is confirmed by the in vitro results showing that nonglycosylated IGFBP-3 is degraded more rapidly in vitro and that its addition to serum results in inhibition of glycosylated IGFBP-3 degradation. Finally, treatment of intact, glycosylated IGFBP-3 in serum with N-glycanase, followed by exposure to the serum protease, resulted in more rapid degradation compared with that of endogenous, glycosylated IGFBP-3 that had not been exposed to N-glycanase. Therefore, the results from multiple different experiments support this conclusion.

Patients with diabetes have been noted to have several changes in the IGF system. Although the changes that occur in patients with type 1 diabetes are relatively reproducible, those that occur in type 2 diabetes are more variable (20, 21). Specifically, studies in type 1 diabetes have shown that patients with poor control have low total serum IGF-I, very low free IGF-I, increases in IGFBP-1 and -2, and decreases in intact IGFBP-3 (22). At least part of the decrease in IGFBP-3 has been attributed to proteolysis. Bereket et al. (13) documented that proteolytic cleavage of IGFBP-3 in vitro was more rapid in serum obtained from children with new-onset type 1 diabetes who had not yet received insulin. Intact IGFBP-3 was estimated to be reduced by 50% compared with normal, control serum. After the initiation of insulin treatment, there was 102 ± 13% increase in intact IGFBP-3. Interestingly, an immunoradiometric assay for IGFBP-3 which detected both intact IGFBP-3 and its fragments, showed only a 30% reduction, and the mean value increased to lesser extent after insulin treatment. The abundance of the major fragment of IGFBP-3 was also noted to be increased in the sera of diabetic patients, and it decreased after the initiation of insulin therapy. When the sera were incubated in vitro with [125I]IGFBP-3, and degradation was measured, it was estimated that sera from untreated diabetics degraded IGFBP-3 128 ± 5% faster compared with control sera, and the degree of proteolysis was decreased to 91 ± 5% of the control value after insulin therapy. The molecular sizes of the fragments detected were similar to those generated by the IGFBP-3 protease in pregnancy serum. They concluded that adequate insulin dosage was a major variable controlling the rate of IGFBP-3 degradation in these patients.

Our study was not designed to determine the effect of glycemic control on IGFBP-3 proteolysis. The findings reported previously (17) had shown significant reductions in fasting glucose and insulin dosage in all four treatment groups, but the greatest decrease in postprandial glucose measurements occurred in the patients in group 3. As shown in Table 1Go, this group had less reduction in the degree of IGFBP-3 proteolysis compared with groups 1 and 2. Although the route of IGFBP-3 administration was different, the changes in the degree of IGFBP-3 proteolysis detected after either 12 h or 14 d of IGF-I/ IGFBP-3 administration between groups 1 and 3 do not appear to be due to improved glycemic control. Furthermore, our in vitro findings strongly suggest that the increase in intact, glycosylated IGFBP-3 that is detected is due to preferential degradation of nonglycosylated IGFBP-3.

The cleavage of IGFBP-3 in patients with type 2 diabetes has been characterized previously. Bang et al. (14) analyzed 23 patients with noninsulin-dependent diabetes mellitus and compared their results to 33 age-matched, nondiabetic subjects. IGFBP-3 values by RIA were found to be decreased approximately 11%. Protease activity, as measured by the rate of disappearance of [125I]IGFBP-3 in vitro, was 154 ± 21% of the control, compared with 84 ± 12% of the control in normal, nonobese adults. The abundance of intact IGFBP-3, as estimated by Western ligand blotting, was 72% of the control, compared with 96% of the control in the normal control subjects. The mean total plasma IGF-I concentration for this group of subjects was –1.0 SD score. Importantly, the investigators correlated the abundance of the intact IGFBP-3 as estimated by Western ligand blotting with RIA values and showed that the RIA consistently overestimated the amount of intact IGFBP-3. This is due primarily to the increase in the 29-kDa fragment of glycosylated IGFBP-3 that was measured in their RIA. These findings strongly suggest that the use of routine RIAs or immunoradiometric assays that measure the 29- to 30-kDa fragment of glycosylated IGFBP-3 will overestimate the amount of intact IGFBP-3 in serum from patients with diabetes and from those patients with other conditions in which extensive IGFBP-3 proteolysis has occurred (14, 23, 24, 25).

The physiological significance of this observation is rendered important by the finding that the affinity of the 30-kDa fragment in solution binding assays for IGF-I and IGF-II is reduced (26). Although this fragment is estimated to have almost no affinity by Western ligand blotting, this is probably not an accurate estimate of its true affinity in serum. However, even when measured by solution binding assays, the reduction in affinity is substantial, and this has led investigators to conclude that IGF-I that is bound to this fragment will equilibrate more rapidly with the extravascular space (27). Although this fragment can bind to the acid-labile subunit (ALS), it is cleared more rapidly by the kidneys, because its abundance is increased in the urine of patients with diabetes, suggesting that its half-life is substantially reduced compared with that of intact IGFBP-3 (28, 29). Furthermore, a reduction in the affinity of this fragment for the IGFs would allow better equilibration with IGFBP-1 and -2. IGFBP-1 may be particularly important in this regard in diabetes, because its plasma concentrations are often elevated. Therefore, after IGFBP-3 proteolysis, a greater percentage of the IGF-I and -II may be bound to IGFBP-1, resulting in more rapid equilibration of the IGFs with the extravascular space (30).

This change in distribution of IGF-I among the various binding proteins has been proposed to act as a compensatory mechanism for the decrease in total IGF-I in diabetic patients who have increased proteolysis of IGFBP-3 in plasma. IGFBP-3 protease activity has been shown to be increased in pregnancy (6, 7, 8), postsurgical catabolic states (9, 10), and severe medical illnesses (11, 12). One common characteristic among all these clinical conditions is the presence of insulin resistance (13, 14). Therefore, IGFBP-3 proteolysis may represent a mechanism by which the bioavailability of circulating IGF-I is increased to counteract excessive catabolism and IGF-I resistance at the tissue level. This rapid proteolytic cleavage of IGFBP-3 with a subsequent decrease in plasma IGF-I no doubt contribute to the reduction in total IGF-I levels, which, although not uniformly observed in type 2 diabetes, is uniform in type 1 diabetes and other clinical conditions noted herein that are associated with increased IGFBP-3 proteolysis.

The 29-kDa fragment of IGFBP-3 noted to be increased in serum has also been found to be increased in the urine of diabetic patients (28, 29, 30). Although it has not been definitively determined whether IGFBP-3 can be further degraded in the kidney, the increased concentration of this fragment in urine could be due to more rapid clearance of the 29-kDa fragment at the level of the glomerulus or more rapid degradation of intact IGFBP-3 in the kidney. Clearly, lower Mr forms of this fragment are generated by the IGFBP-3 protease in vitro as shown in Fig. 4Go. However the very low levels of these fragments in sera from diabetic patients compared with the abundance of the 29-kDa fragment suggest that they are cleared more rapidly from the vascular compartment. Analysis of urine from diabetic patients by immunoblotting has shown that many of these fragments are detectible (29, 30). Although it cannot be excluded that the kidney itself is generating these fragments, this data combined with the results of serum analyses suggest that many of these fragments are cleared intact, filtered into the urine, then excreted. The reason for the prolonged half-life of the 29-kDa fragment compared with these lower Mr fragments, probably relates to its ability to bind to ALS (31, 32). However, some of the 29-kDa fragment that is present in diabetic serum is probably cleared by the kidney, because it does not bind to IGF-I as avidly, and therefore, less IGF-I/29-kDa binary complex would associate with the ALS to form ternary complexes (this is supported by the abundance of the 29-kDa fragment in diabetic urine compared with nondiabetic urine). However, the relative abundance of this fragment in serum suggests that at least a portion of this material forms a stable complex with IGF-I or -II and ALS. Thus, some IGF-I-carrying capacity of this fragment is preserved in diabetic serum. Whether the IGF-I bound to this fragment is truly more bioavailable than that bound to intact IGFBP-3 has not been definitively determined, but the difference in the affinity of this fragment compared with that of intact IGFBP-3 in solution binding assays would suggest that this is the case.

Our data suggest that exogenous administration of nonglycosylated IGFBP-3 is an important variable for controlling proteolysis of endogenous, glycosylated IGFBP-3. Peak intact IGFBP-3 levels obtained 14 d after treatment at a time when there was substantial inhibition of degradation of nonglycosylated IGFBP-3 were significantly greater than the baseline values. Furthermore, the group that had the highest nonglycosylated IGFBP-3 concentration showed the greatest preservation of glycosylated IGFBP-3, i.e. group 2. This group also had the highest total IGF-I and the highest free IGF-I concentrations, suggesting that the free IGF-I is in equilibrium with IGF-I that is bound to intact IGFBP-3, and the greater the concentration of intact IGFBP-3 and total IGF-I, the greater the reservoir available to generate free IGF-I. This also correlates with the biological response, because these groups had the greatest reduction in endogenous insulin dose and the greatest reduction in fasting glucose (17). Thus, the degree of improvement in insulin sensitivity may correlate with the attenuation of IGFBP-3 proteolysis that occurs in response to administration of nonglycosylated IGFBP-3, and this may contribute to the enhancement of IGF-I bioavailability and thus the improvement in insulin sensitivity.

The mechanism by which IGFBP-3 is degraded has not been entirely elucidated; however, Maile et al. (15) demonstrated that an inhibitor of IGFBP-3 proteolysis was present in normal serum and that it protected IGFBP-3. The addition of heparin to normal serum induced IGFBP-3 protease activity to a level comparable to that in pregnancy serum, suggesting that the same concentrations of the protease are present in normal and pregnancy sera and that the differences among pregnancy, diabetic, and normal serum are due to the amount of this inhibitor present. They also demonstrated that this protease activity degraded nonglycosylated IGFBP-3 preferentially in vitro. Lassarre et al. (33) found a strong relationship between the degree of IGFBP-3 proteolysis in the serum of diabetic patients and hemoglobin A1C, suggesting that the amount of protease activity was related to diabetic control. They concluded that this change in the amount of IGFBP-3 proteolysis was a compensatory mechanism in diabetes, occurring as an adaptation to insulin deficiency or inadequate insulin action. In contrast, in disorders of GH secretion, this compensation could not be shown, and the degree of IGFBP-3 proteolysis did not correlate with the degree of IGF-I deficiency or excess.

The identity of the IGFBP-3 protease activity in diabetic serum has not been definitively determined. Most studies have suggested that it is a divalent cation-dependent serine protease (6, 7). Several candidate serine proteases, such as a plasmin (34), prostate-specific antigen (35), and kallikreins 2 and 3 (36) are present in serum and degrade IGFBP-3. However, metalloproteases such as Adam 12 also degrade IGFBP-3 (37), and definitive proof that any of these accounts for the protease activity in diabetic serum has not been forthcoming. The fact that insulin can induce plasminogen inactivator inhibitor-1 synthesis suggests that plasmin could contribute to this activity (38). However, the IGFBP-3 fragments generated by plasmin differ in molecular size from those generated by incubation of IGFBP-3 with pregnancy or diabetic serum.

In summary, diabetic serum contains increased proteolytic activity for IGFBP-3 that is probably due to reduced amounts of a protease inhibitor. This leads to rapid cleavage of exogenously administered, nonglycosylated IGFBP-3. Administration of high concentrations of nonglycosylated IGFBP-3 functions to reduce the degradation of endogenous, glycosylated IGFBP-3, thus leading to enhanced stability of IGF-I in serum. This enhanced stability may contribute to the ability of the IGFBP-3/IGF-I complex to enhance insulin sensitivity in patients with diabetes. Thus, the dose and mode of delivery of nonglycosylated IGFBP-3 may be variables in determining the rate of degradation of glycosylated IGFBP-3 and thus indirectly in controlling IGF-I bioavailability.


   Acknowledgments
 
We thank Ms. Laura Lindsey for her help with preparing the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grant AG-02331.

First Published Online September 27, 2005

Abbreviations: ALS, Acid-labile subunit; IGFBP-3, IGF-binding protein-3; Mr, molecular mass.

Received June 22, 2005.

Accepted September 14, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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D. R. Clemmons, M. Sleevi, G. Allan, and A. Sommer
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