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Medical Research Laboratories (J.F., K.N.R., S.P.J., H.Ø.), Aarhus University Hospital, DK-8000 Aarhus C, Denmark; and Department of Endocrinology (K.H., M.W.-C.), Odense University Hospital, DK-5000 Odense C, Denmark
Address all correspondence and requests for reprints to: Dr. Jan Frystyk, Institute of Experimental Clinical Research, Aarhus Kommune Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. E-mail: jan{at}frystyk.dk
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Abstract |
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r 0.98; P < 0.0001). Free IGF-I correlated inversely with IGFBP-1 (-0.81 r -0.48; 0.0001 P 0.05) and the binary complex (-0.79 r -0.41; 0.0001 P 0.05). To study overnight fasting levels, we compared healthy controls and patients with type 1 diabetes and chronic renal failure (n = 10), because these patients show profound alterations in their IGF-system. In both groups, the binary complex was increased about 2.5-fold (P < 0.0001), whereas IGFBP-1 was increased by 5- to 6-fold (P < 0.0001). Accordingly, free IGF-I was severely reduced (P < 0.0001). In conclusion, the assay enables us to study the role of IGFBP-1 as a dynamic regulator of free IGF-I. Our results clearly show that IGFBP-1 and free IGF-I are tightly associated peptides. Furthermore, it has now become possible to compare levels of IGF-I carried within the binary complex IGFBP-1:IGF-I in different (patho-) physiological conditions. |
Introduction |
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IGFBP-1 is controlled primarily by portal insulin, which rapidly suppresses the hepatic IGFBP-1 synthesis and secretion. Based on the inverse relationship between insulin and IGFBP-1 and the inhibitory effect of IGFBP-1 on IGF-I-mediated actions, IGFBP-1 was suggested to act as a linkage between nutrition and growth. Supportive of this view, levels of IGFBP-1 are up-regulated in conditions characterized by catabolism, i.e. severe infectious diseases, chronic renal failure (CRF), type 1 diabetes, and long-term fasting, among others (2).
On a molar basis, IGFBP-1 constitutes only a few percent of the total pool of circulating IGFBP (3); and accordingly, changes in IGFBP-1 have only little impact on serum levels of total (extractable) IGF-I. However, several clinical investigations have suggested that IGFBP-1 is one of the most important regulators of free IGF-I in vivo (4, 5).
The opposite directed changes of free IGF-I and IGFBP-1, and the inhibitory actions of IGFBP-1 on IGF-I-mediated effects, provide strong (albeit indirect) evidence that IGFBP-1 affects IGF-I bioactivity in vivo by regulating levels of free IGF-I. To test this, we developed a novel time-resolved immunofluorometric assay for the binary complex of IGF-I and IGFBP-1 in human serum. Thus, we have a new tool to study the possible role of IGFBP-1 as a dynamic regulator of circulating free IGF-I in vivo. Furthermore, the assay allows us to compare the level of IGF-I carried within the binary complex IGFBP-1:IGF-I in different physiological and pathophysiological circumstances.
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Materials and Methods |
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A monoclonal human (h)IGFBP-1 antibody (MAB 6303, obtained from Medix Biochemica, Kauniainen, Finland) was used for coating. This antibody was selected because it recognizes several different isoforms of hIGFBP-1 (6). An europium-labeled monoclonal hIGF-I antibody (DSL Inc., Webster, TX) served as 2nd antibody. This antibody is also used in our in-house IGF-I time-resolved immunofluorometric assay as previously described (7). Purified amniotic IGFBP-1 was obtained from HyTest Ltd. (Turku, Finland); recombinant (r)hIGFBP-2, -3, and -4 from R \|[amp ]\| D Systems (Oxon, UK); and rhIGF-I and -II from Austral Biologicals (San Ramon, CA).
Buffers
Coating buffer was made of 15 mM sodium carbonate, 35 mM sodium hydrogen carbonate, pH 9.6. Blocking buffer contained 1.0% (wt/vol) human serum albumin (HSA) (Behring AG, Marburg, Germany) dissolved in 40 mM phosphate, 0.05% (wt/vol) NaN3, 9 g/liter NaCl, and 1.6 g/liter Titriplex V, pH 8.0. IGFBP-1 was dissolved in an in-house assay buffer made of 40 mM phosphate (pH 8.0), 2 g/liter HSA (Behring AG), 9 g/liter NaCl, 2% (vol/vol) Tween 20, 1.6 g/liter Titriplex V, and 0.05% (wt/vol) NaN3. Standards and serum samples were dissolved in Krebs Ringer bicarbonate buffer adjusted to pH 7.4 with CO2 and added 50 g/liter HSA (Behring AG). Wash solution was made of 50 mM Tris-HCl, 9 g/liter NaCl, 0.5% (vol/vol) Tween 20, and 0.05% (wt/vol) NaN3, pH 8.0. Before the plates can be read in a time-resolved plate fluorometer, the europium ions need to be dissociated from solid-phase bound antibodies and brought into solution. This was done by use of the commercial Enhancement Solution (Wallac Oy, Turku, Finland) (8).
Assay principle
The equilibrium between free and bound IGF-I is highly dependent on temperature and buffer constitution (9); and therefore, the assay was performed at conditions approaching those in vivo. Thus, before assay, all serum samples were equilibrated in a water bath at 37 C, and this temperature was maintained during antigen incubation. Furthermore, all samples (standards and sera) were diluted in Krebs Ringer bicarbonate buffer. The assay was performed in 96-well microtest-plates (Wallac Oy). Coating was performed overnight at 5 C, using 250 µl hIGFBP-1 antibody (2.5 mg/liter) per well. Blocking buffer (300 µl per well) was incubated for 3 h at room temperature or overnight at 5 C. After blocking, the wells used for standards received 200 µl IGFBP-1 (100 µg/liter) dissolved in assay buffer, whereas wells used for unknown samples received buffer only. The plates were sealed and incubated overnight at 5 C. Next day, after washing, wells containing immobilized IGFBP-1 were incubated with a serial dilution of IGF-I ranging from 2–1000 pM, whereas wells without IGFBP-1 were used for unknown samples. The molar range of the IGF-I standards equals a serial dilution of IGFBP-1 ranging from 0.06–30 µg/liter, and we decided to use this range because the concentrations of binary complex were going to be compared with levels of IGFBP-1 rather than with IGF-I. All standards and samples (150 µl) were analyzed in duplicate, whereas nonspecific binding (NSB) was analyzed in quadruplicate. After addition of standards and samples, plates were covered with film and incubated on a plate shaker at 37 C. After antigen incubation, all wells were aspirated, washed six times, and incubated with 200 µl europium labeled IGF-I antibody dissolved in assay buffer. Plates were incubated overnight at 5 C, washed six times, and incubated with 200 µl per well Enhancement Solution (Wallac Oy), and read in a time-resolved fluorometer.
Assay validation was performed by use of nonfasting serum samples obtained from healthy subjects (n = 6). The assay was validated in several different ways: 1) Parallelism between standards and samples was examined using serial dilutions of serum. 2) To study whether it was necessary to equilibrate serum at 37 C before assay, samples were incubated either at room temperature or in a water bath at 37 C for 10, 20, 30, 40, and 60 min. 3) Antigen incubation time was examined by incubating standards and samples for 1, 2, and 3 h. 4) Within and in-between coefficients of variation (CVs) were estimated by repetitive measurements of the samples. 5) Repetitive freezing and thawing of serum were used to examine the stability of the binary complex. 6) To study the effect of exogenous IGFBP-1 and IGF-I, respectively, on binary complex formation, sera were incubated overnight at 5 C with a serial dilution of amniotic IGFBP-1 (0, 62.5, 125, 250, 500, and 1000 µg/liter) or IGF-I (0, 12.5, 25 50, 100, and 200 µg/liter) before assay. 7) Assay recovery of binary complexes was examined in buffer as well as in serum. The recovery of IGFBP-1 was investigated by incubating a serial dilution of IGFBP-1 (2.5–40 µg/liter) in MAB 6303-coated wells. After 2.5 h of incubation, samples were aspirated and analyzed for IGFBP-1 immunoreactivity. The recovery was calculated as the difference between the concentration of IGFBP-1 in the sample before and after incubation. Similarly, the recovery of a serial dilution of IGF-I (from 15–1000 pM) was estimated using wells with MAB 6303-bound IGFBP-1. The recovery of binary complex in serum was estimated by incubating six control samples as described for IGFBP-1. 8) Assay specificity was examined in different ways. First, we studied the effect of omitting one of the two components of the binary complex. A serial dilution of IGFBP-1 (0, 250, 500, and 1000 µg/liter) was incubated in MAB 6303-coated wells, followed by incubation of europium labeled IGF-I antibody (omitting IGF-I). Similarly, a serial dilution of IGF-I (0, 250, 500, and 1000 µg/liter) was incubated in MAB 6303-coated wells, followed by incubation of europium-labeled antibody (omitting IGFBP-1). Second, we incubated a serial dilution of IGF-II (0, 250, 500, and 1000 µg/liter) in wells containing MAB 6303-bound IGFBP-1, followed by addition of europium-labeled IGF-I antibody. Because IGF-II may not only interfere by simple cross-reaction but also by affecting the existing equilibrium between IGF-I and IGFBP-I, we studied the effect of exogenous IGF-II on IGFBP-1:IGF-I complex formation in serum. This study was performed as described for IGF-I. Third, the cross-reactivity of rhIGFBP-2, -3, and -4 were examined by incubating serial dilutions (0, 500, 1000, 2500, 5000, and 10000 µg/liter) in wells coated with MAB 6303 in the absence or presence of IGF-I (3.85 µg/liter, 
500 pM).
Clinical evaluation
To study the dynamic relationship between free IGF-I, IGFBP-1, and the binary complex, we examined seven healthy males [age = 31.3 ± 2.6 yr; body mass index (BMI) = 24.2 ± 1.2 kg/m2] during 72 h of fasting. The participants began fasting at home, from 2100 h. Next morning, they were admitted to a hospital, and they remained there for the next 72 h. Serum samples were collected every 3 h during admittance. The participants had free access to water, but no food was allowed, and compliance with the fast was assessed by determination of serum free fatty acids, glucose, and insulin. None of the participants developed hypoglycemia during the fast.
Type 1 diabetes and CRF are both characterized by elevated serum IGFBP-1 and reduced free IGF-I (10, 11); and therefore, we found it of interest to determine the concentration of binary complex in these patient groups. Overnight fasting serum samples were collected from three groups of age- and BMI-matched males (n = 10): healthy controls (age = 45 ± 3 yr, BMI = 25.9 ± 1.3 kg/m2, glomerular filtration rate (GFR) = 153 ± 12 ml/min·1.73m2, HbA1c = 5.3 ± 0.1%), patients with type 1 diabetes (age = 48 ± 3 yr, BMI = 25.1 ± 0.7 kg/m2, HbA1c = 8.4 ± 0.4%), and nondiabetic patients with CRF (age = 49 ± 3 yr, BMI = 24.8 ± 1.2 kg/m2, GFR = 27 ± 4 ml/min·1.73m2). All blood samples were obtained in accordance with the declaration of Helsinki.
Other assays used
Ultrafiltered free and total extractable IGF-I and -II were determined by in-house assays as previously described (7, 9). Commercial assays were used for determination of IGFBP-1, IGFBP-3 (DSL, Inc.), and GH (Wallac Oy).
Statistics
Parametric data were compared by repeated-measures ANOVA or one-way ANOVA, whereas nonparametric data were compared by Friedman’s repeated-measures ANOVA on ranks or Kruskal-Wallis’ one-way ANOVA on ranks. A significant ANOVA was followed by Student Newman Keul’s test for multiple comparisons. In the fasting study, we compared the area under curve (AUC) for each of the three 24-h periods. Linear regression analysis was used to study the relationship between free IGF-I, IGFBP-1, and the binary complex. P-values < 0.05 were considered statistically significant.
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Results |
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A representative standard curve is shown in Fig. 1
. The CVs of the standards averaged less than 5%, and the detecting limit (NSB plus 3 SD) was estimated to be less than 0.5 µg/liter. Serum measurements diluted in parallel with the standard curve (Fig. 1). However, in diabetic serum with elevated levels of IGFBP-1, the assay became saturated at low serum dilutions.
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Addition of IGF-I to serum increased binary complex formation (P < 0.0001; Fig. 2, upper panel). However, at high IGF-I concentrations, binary complex formation leveled off, and there was no difference when comparing 50, 100, and 200 µg/liter IGF-I. In contrast, addition of IGF-II decreased the signal in a dose-dependent manner (P < 0.0001; Fig. 2, lower panel). Addition of IGFBP-1 resulted in a biphasic response (P < 0.0001; Fig. 2, lower panel): low concentrations of IGFBP-1 increased binary complex formation, whereas higher concentrations caused a reduction toward baseline.
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Clinical evaluation
Free IGF-I and -II responded similarly to fasting (Fig. 3): both free fractions were reduced by approximately two thirds (P < 0.0001), and levels correlated positively in all seven participants (0.56 r 0.85; 0.0001 P 0.004). Concomitantly, a parallel increase in IGFBP-1 and the binary complex was observed (P < 0.0001), i.e. the fraction of IGFBP-1 carrying IGF-I remained unchanged during the study period, with an overall mean of 37.7 ± 0.6%. In all participants, we observed highly significant correlations among free IGF-I, IGFBP-1, and the binary complex (Table 1), as well as between free IGF-II and IGFBP-1 (-0.79 r -0.44; 0.0001 P 0.03). Total IGF-II and IGFBP-3 remained unchanged throughout, whereas total IGF-I decreased marginally (but significantly) during the third day of fasting (P < 0.0001; Fig. 4). Twenty-four-hour GH secretion was significantly increased at d 2, when compared with d 1 and d 3 (P < 0.02; Fig. 4).
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). In patients with type 1 diabetes and CRF, levels of binary complex were increased about 2.5-fold (P < 0.0001). However, because levels of IGFBP-1 were increased 5- to 6-fold (P < 0.0001), the fraction of IGFBP-1 saturated with IGF-I was relatively reduced in both patient groups (P < 0.0001). Finally, serum free IGF-I was reduced by 60–80% in the two patient groups (P < 0.0001), whereas total IGF-I was significantly reduced in CRF only (P < 0.02).
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Discussion |
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In the present assay, we used MAB 6303 (obtained from Medix Biochemica) to capture the binary complex of IGF-I and IGFBP-1. This monoclonal antibody was selected because it has previously been shown to recognize the predominant form of IGFBP-1, which is highly phosphorylated and constitutes about 90% of the circulating IGFBP-1 pool, as well as less- and nonphosphorylated isoforms of IGFBP-1 (6). However, because the binding characteristics of MAB 6303 were based on its ability to immunoprecipitate IGFBP-1 (6), we cannot exclude that there may exist minor circulating isoforms, which are not recognized by the antibody, and therefore remains undetected by our assay. On the other hand, with the exception of pregnancy, during which the level of less- and nonphosphorylated isoforms becomes markedly increased, there has been no report on major changes in the phosphorylation pattern of IGFBP-1 (6, 12). Thus, we believe the possible failure of the antibody to recognize all isoforms of hIGFBP-1 may result in minor errors only, at least in nonpregnancy samples.
The degree of phosphorylation of IGFBP-1 is known to affect the affinity for IGF-I (13); and, in theory, this may alter the affinity for MAB 6303 as well. However, by using a noncompetitive assay model, we were able to circumvent this possible pitfall. In a noncompetitive assay, the coating antibody always remains in molar excess of the antigen; and therefore, virtually all immunoreactive antigens become captured. In contrast, if we had chosen a competitive assay model, in which an excess of antigens compete for binding with MAB 6303, differences in binding affinity might have caused problems.
So far, the hypothesis that free IGF-I and IGFBP-1 are inversely associated peptides has been based on correlation studies (4, 5, 10, 11), which (at best) yield indirect evidence only of a physiological relationship. Furthermore, previous studies have made it clear that the observed changes in IGFBP-1, on a molar basis, by far exceed those of free IGF-I (4, 5). Therefore, we found it of interest to develop a method that enabled us to further explore the dynamic relationship between free IGF-I and IGFBP-1, and we found that this could be achieved by measuring complex formation between the two peptides.
In accordance with previous findings, fasting induced opposite directed changes in levels of free IGF-I and IGFBP-1, supporting the physiological association between the two peptides (5). Now, we are able to show that the increase in IGFBP-1 is accompanied by increased levels of binary complex, which furthermore correlated positively with IGFBP-1 and inversely with free IGF-I. Thus, our findings yield strong evidence that the reduction in free IGF-I is explained by increased complex formation with IGFBP-1. On the other hand, it is clear that the increase in binary complex (about 48 µg/liter, 12 µg/liter of IGF-I), observed during 72 h of fasting, by far exceeded the reduction in free IGF-I (0.3 µg/liter). Thus, there must be another source of IGF-I for binary complex formation in vivo. The present in vitro studies showed that addition of IGFBP-1, up to about 125 µg/liter, dose-dependently increased binary complex formation (Fig. 2
, lower panel). In these samples, the baseline level of free IGF-I averaged approximately 1 µg/liter; and therefore, the amount of IGF-I necessary for the observed in vitro formation of binary complex must originate from the other IGFBPs. Therefore, our study strongly indicates that the interaction between IGF-I and the IGFBPs is a highly dynamic process in vitro as well as in vivo. However, in vivo, another possibility coexists: Lewitt et al. (14) studied the effect of excess IGFBP-3 on ternary complex formation (composed of IGF-I, IGFBP-3, and the acid labile subunit). In rats, iv administration of IGFBP-3 resulted in rapid formation of ternary complexes, whereas this was not observed in vitro. Because IGFBP-3 must bind IGF-I before it can associate with the acid labile subunit, the authors suggested that the availability of IGF-I was much greater than previously expected. Thus, in theory, part of the IGF-I needed for binary complex formation in vivo could originate from a pool of readily available IGF-I present in extravascular tissues. However, this issue remains to be clarified.
Previous investigations have shown that total IGF-I is more sensitive than total IGF-II to short-term fasting (15), and this was confirmed in the present study. Still, free IGF-I and -II were equally suppressed by fasting, and this confirms that free IGF is far more sensitive, than total IGF, to nutritional changes (5). In this context, we find it noteworthy that the increase in GH secretion was preceded by a reduction in free (but not total) IGF-I. Thus, our study supports the hypothesis that the pituitary GH secretion is feedback-controlled, in part, by circulating levels of free (rather than total) IGF-I (16).
As for free IGF-I, the reduction in free IGF-II is likely to be explained by an up-regulation of IGFBP-1. This view is supported by the strong inverse correlation between free IGF-II and IGFBP-1 observed in all seven subjects. Because IGFBP-1 seems to regulate free IGF-II, it was obvious to modify our assay to include determination of IGFBP-1 complexed IGF-II. However, we were unable to find a suitable IGF-II antibody, and we speculate that it may be because of different spatial conformations of the two binary complexes. Thus, we cannot estimate the total saturation of IGFBP-1 but only the fraction of IGFBP-1 carrying IGF-I.
As previously observed, patients with type 1 diabetes and CRF showed markedly reduced levels of free IGF-I (10, 11). This finding may be partly explained by the concomitant increase in serum IGFBP-1, which correlates inversely with free IGF-I in both patient groups (10, 17). Accordingly, the overnight fasting levels of binary complex were increased about 2.5-fold in type 1 diabetes and CRF, compared with controls. Because IGFBP-1 most likely inhibits IGF-I-mediated effects by controlling interaction between the growth factor and its cellular receptors, this finding indicates that more IGF-I is inactivated in CRF and type 1 diabetes than in the normal state. In addition, IGFBP-1 was elevated 5- to 6-fold. Thus, the fraction of IGFBP-1 carrying IGF-I was reduced about 50%, compared with controls. This finding implies that more IGFBP-1 remains unsaturated in type 1 diabetes and CRF than in normal subjects. In this context, we find it worth noting that, in CRF, the reduced renal clearance results in an accumulation of unsaturated IGFBPs (IGFBP-1 among others), which are believed to play a pivotal role in the growth retardation seen in children with CRF (18).
In conclusion, we have developed a highly specific and sensitive assay for the binary complex of IGFBP-1-bound IGF-I in human serum. The novel assay has enabled us to study both the dynamic relationship between IGFBP-1 and free IGF-I as well as the impact of chronically elevated IGFBP-1 on free IGF-I levels. Our short-term fasting study yields strong evidence that IGFBP-1 and free IGF-I are closely inversely associated peptides. Furthermore, we show that, in patients suffering from type 1 diabetes and CRF, the amount of IGF-I carried within the binary complex is markedly elevated. The latter finding supports the idea that IGFBP-I may act as an inhibitor of IGF-I actions by blocking the interaction between IGF-I and the cellular IGF-I receptors.
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Acknowledgments |
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Footnotes |
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Abbreviations: AUC, Area under curve; BMI, body mass index; CRF, chronic renal failure; CV, coefficient of variation; hIGF, human IGF; HSA, human serum albumin; IGFBP, IGF-binding protein; NSB, nonspecific binding; rhIGF, recombinant human IGF.
Received April 13, 2001.
Accepted October 1, 2001.
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) and a patient with type 1 diabetes (
). The molar range of the IGF-I standards equals a serial dilution of IGFBP-1, ranging from 0.06–30 µg/liter, and we decided to use this range because the concentrations of binary complex were going to be compared with levels of IGFBP-1 rather than IGF-I. Only mean values are shown because the size of the error bars did only occasionally exceed the size of the symbols. CPS, Counts per second.
; upper panel), IGF-II (
, P < 0.05, compared with baseline (no addition).
; lower panel), and the binary complex (
; lower panel) during 72 h fasting. The dashed lines indicate the three 24-h periods that were used for calculation of AUCs. 
