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Medical Research Laboratories (C.S., A.K., H.Ø., A.F.), Institute of Experimental Clinical Research, Aarhus University; and Medical Department M (J.M., N.V., J.W., A.F.), Aarhus Kommunehospital, DK-8000 Aarhus, Denmark
Address all correspondence and requests for reprints to: Christian Skjærbæk, Medical Research Laboratories, Building 3, Aarhus Kommunehospital, 8000 Aarhus C, Denmark.
Abstract
Increased proteolysis of insulin-like growth factor binding protein (IGFBP)-3 is seen in several pathophysiological conditions and may represent an important mechanism for the regulation of insulin-like growth factor bioavailability. It has previously been suggested that proteolysis of IGFBP-3 is dependent on the GH status. To investigate this, IGFBP-3 proteolysis was measured in three groups of subjects: 1) GH-deficient patients before and after GH replacement (n = 14); 2) healthy subjects before and after 14 days of GH administration (n = 7); and 3) acromegalic patients before and after treatment with a long-acting SRIH analogue (octreotide; n = 14). In vivo IGFBP-3 proteolysis was investigated by Western immunoblotting. No difference was detected in pretreatment samples, and GH treatment in GH-deficient subjects or octreotide treatment in acromegalic subjects had no impact on in vivo proteolysis. In contrast, GH administration to healthy subjects caused a 21% increase in in vivo proteolysis (P = 0.0008). In vitro IGFBP-3 proteolysis was investigated by incubation of serum with 125I-rhIGFBP-3, followed by SDS-PAGE. In pretreatment samples, the percentage of proteolyzed 125I-rhIGFBP-3 was 13 ± 1% (acromegalic subjects), 11 ± 1% (healthy subjects), and 9 ± 1% (GH-deficient subjects) (P < 0.009, GH-deficient vs. acromegalic subjects). Treatment had no effect on in vitro proteolysis. We conclude that GH status has no major impact on IGFBP-3 protease activity in serum.
THE INSULIN-LIKE growth factor binding proteins (IGFBPs) contain six peptides (IGFBP-1 through IGFBP-6) (1, 2) that have high affinities to insulin-like growth factors I and II (IGF-I and -II) and probably contain several other peptides with lower affinity (3). The IGFBPs are believed to modulate the growth promoting and metabolic actions of the IGFs. About 80% of circulating IGFs are complexed with IGFBP-3 and acid-labile subunit, forming a high molecular mass (150 kDa) ternary complex (4).
The complexity of this system has been further increased by the demonstration of specific, yet largely unidentified, proteases that cleave IGFBPs. Proteolysis of IGFBP-3 was initially observed in pregnancy serum (5, 6) but has now been described in several pathophysiological conditions, such as Laron-type dwarfism (7), critical illness of various kinds (8), insulin-dependent diabetes mellitus (9), non-insulin-dependent diabetes mellitus (10), and after major surgery (11); but it is found also in serum from healthy, nonpregnant subjects (12, 13). The cleaved fragments fail to bind IGFs on Western ligand blots (WLBs) and apparently do have a decreased affinity for IGFs (5, 14). The physiological significance of the IGFBP-3 proteolysis remains unknown, but the decreased affinity of the fragments for IGFs may increase IGF availability to tissues and cells and thereby increase IGF bioactivity (15, 16)
GH plays a major role in the regulation of the IGF system at several levels, GH being the principal regulator of all three components of the 150-kDa ternary complex. Furthermore, a role for GH in the regulation of IGFBP-3 proteolysis also has been suggested. Lassarre et al. (17) found, by Western immunoblotting (WIB), that the amount of proteolyzed IGFBP-3 was increased in GH deficiency (GHD) and decreased in acromegaly, when compared with normal subjects. Contrasting results were reported by Rutishauser et al. (18), who found decreased IGFBP-3 protease activity in hypophysectomized rats with normalization after GH substitution. Fielder et al. (19) found no effect of hypophysectomy on IGFBP-3 proteolysis in either virgin or pregnant mice. Apart from the obvious difference in species, the divergency could be caused by different methodologies. Rutishauser et al. (18) and Fielder et al. (19) used the method of Lamson et al. (20), incubating serum with 125I-rhIGFBP-3. This method determines the proteolytic activity present in vitro, whereas WIB visualizes the proteolysis that has already taken place in vivo. However, none of the previous studies have examined changes in IGFBP-3 protease activity in the same subjects in response to GH administration or suppression of endogenous GH secretion.
Accordingly, the present study examined both the in vivo and the in vitro IGFBP-3 proteolysis in serum from subjects with different GH status and, in addition, the response to GH treatment and GH suppression.
Materials and Methods
Subjects
All serum samples were obtained after an overnight fast. Serum was obtained from 3 groups of subjects/patients: 1) a group of 14 patients with adult onset GHD (age: 47 ± 2 yr; body mass index (BMI): 28 ± 1 kg/m2; male/female: 9/5) before and after 1 yr of treatment with GH, 2 IU/m2 (Norditropin, Novo Nordisk, Bagsværd, Denmark); 2) a group of 7 healthy subjects (age: 27 ± 1 yr; BMI: 23 ± 1 kg/m2; male/female 7/0) before and after 14 days of treatment with GH, 6 IU/m2 (Norditropin, Novo Nordisk) and 3) a group of 14 acromegalic patients (age: 50 ± 4 yr; BMI: 28 ± 1 kg/m2; male/female: 6/8) before and after 12 months of treatment with a microsphere-encapsulated, slow-release preparation of octreotide (Sandostatin LAR, Sandoz, Basle, Switzerland). All patients were well controlled on octreotide, based on the available clinical and biochemical data. All samples were obtained after informed consent, and the study was performed in accordance with the declaration of Helsinki, and after approval of the regional ethics committee.
WLB and WIB for serum IGFBP-3
SDS-PAGE and ligand blot analysis of all samples were performed according to the method of Hossenlopp et al. (21), as previously described (22). Two microliters of serum was subjected to SDS-PAGE (10% polyacrylamide) under nonreducing conditions. Samples from each subject/patient were analyzed on the same gel. IGFBP-3 immunoblot analysis was performed on all samples with a polyclonal IGFBP-3 antibody (1:1000; Upstate Biotechnology Incorporated, Lake Placid, NY) with 35S-protein A (SA = 500 Ci/mmol; Amersham International, Amersham, UK).
Quantification of WIB, WLB, and IGFBP-3 degradation assay
Autoradiograms of WLBs, WIBs, and the 125I-IGFBP-3 degradation assay were quantified by densitometry using a Shimadzu CS-9001 PC dual-wavelength flying spot scanner. The relative density of the bands was measured as arbitrary absorbance units per square millimeter (AU/mm2).
IGFBP-3 degradation assay
The IGFBP-3 protease assay was performed on all samples, as previously described (20), using a preparation of recombinant human 125I-rhIGFBP-3 without enzyme-inhibiting additives (Diagnostic Systems Laboratories, Webster, TX). 125I-IGFBP-3 (approximately 30,000 cpm) was incubated for 18 h at 37 C with 2 µL of serum samples from subjects/patients and subjected to SDS-PAGE, as described above. On each gel, internal control sera from normal controls and term-pregnant subjects were included. Electrophoresed gels were fixed in a 7% acetic acid solution, dried, and autoradiographed. The amount of proteolysis was calculated as a ratio of the absorbance of fragmentated 125I-IGFBP-3 divided by the sum of all 125I-IGFBP-3 (38–42, 30, and 16–18 kDa) related optical densities in that lane and expressed as a percentage (in vitro proteolysis). In the same way, IGFBP-3 proteolysis (in vivo proteolysis) was calculated from WIBs as a ratio of fragmented IGFBP-3 (30 and 16–18 kDa) divided by the sum of all IGFBP-3 (38–42, 30, and 16–18 kDa) in each lane.
IGFBP-3 immunoassay
Serum IGFBP-3 was measured in all samples by a commercial immunoradiometric assay (IRMA; Diagnostic Systems Laboratories), calibrated to recombinant nonglycosylated human IGFBP-3 (29 kDa). The same polyclonal antibody was used as catching antibody and detection antibody. All samples were run in the same assay.
Statistics
Results are given as mean ± SEM. One-way ANOVA was used to test differences in pretreatment values among the three groups. Student’s t test for paired data was used to evaluate changes to treatment within the groups. A P-value of less than 0.05 was considered significant.
Results
Total IGFBP-3
The total amount of IGFBP-3 in all samples was determined in three
ways; by WIB (by summing the area of all bands), by WLB, and by IRMA.
Representative blots of WIB and WLB are shown in Fig. 1
. Three distinct bands appeared on the
WIBs: a doublet of 38–42 kDa (representing intact IGFBP-3 of various
glycosylation) and bands of 30 and 16–18 kDa (representing fragments
of IGFBP-3). Results of densitometric analysis are shown in Fig. 2. The WLB showed four distinct bands, of
which the 38- to 42-kDa doublet represents IGFBP-3. Fig. 3 shows the results of all three IGFBP-3
analyses. By IRMA, acromegalic patients had increased levels of IGFBP-3
before treatment, when compared both with normal subjects and with GHD
patients, whereas there was no significant difference between the two
latter groups. GH treatment caused an increase in IGFBP-3 both in
normal subjects and in GHD patients, and treatment with octreotide
caused a decrease in IGFBP-3 in the acromegalic group. The same
differences were detected by WIB, with the notable exception that
pretreatment levels in acromegalic patients were significantly
different only from those of GHD patients. Pretreatment levels of
IGFBP-3 in acromegalic patients by WLB were increased when compared
with GHD, and IGFBP-3 increased in the GHD group after GH
treatment.
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Results are shown (see Fig. 5, upper panel).
Pretreatment levels of proteolysis were 34 ± 2% (acromegalic
patients), 30 ± 2% (healthy subjects), and 36 ± 3% (GHD
patients), with no significant differences. Treatment with octreotide
in acromegalic patients and treatment with GH in GHD patients caused no
significant changes in in vivo IGFBP-3 proteolysis, whereas
treatment with GH in healthy subjects caused a 21% increase in the
in vivo proteolysis (P = 0.0008).
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A representative 125I-IGFBP-3 degradation
autoradiograph is shown in Fig. 4. The
results of the IGFBP-3 protease assay are shown in Fig. 5
, lower panel. The
percentages of proteolyzed 125I-IGFBP-3 in pretreatment
samples of the three groups were 13 ± 1% (acromegalic patients),
11 ± 1% (healthy subjects), and 9 ± 1% (GHD patients).
The protease activity in acromegalic serum was 31% higher than that of
GHD (P = 0.009) and tended to be higher than in healthy
subjects (P = 0.07). However, treatment with GH in
healthy subjects, and GHD and treatment with octreotide in acromegalic
patients, did not alter protease activity.
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In the present study, we investigated the effect of GH on IGFBP-3 proteolytic activity in human serum using two different protease assays: a direct 125I-IGFBP-3 degradation assay for in vitro proteolysis, and WIB with a specific antibody for in vivo proteolysis. In brief, we found neither any systematic relationship between GH status and serum IGFBP-3 proteolytic activity by any of the protease assays, nor any major effect of GH treatment or GH suppression.
Since the first reports of serum IGFBP-3 proteolysis in pregnancy (5, 6), the phenomenon has been described in several pathophysiological
conditions but has been shown to occur also in serum from healthy,
nonpregnant subjects (12, 13). Although several peptides, such as nerve
growth factor (23), prostata specific antigen (23, 24), and plasminogen
(25), have been shown to possess proteolytic activity against
IGFBP-3 in vitro, the true nature and regulation of the
proteases demonstrated in vivo are largely unidentified.
Recently, Bereket et al. (9) suggested a key role for
insulin, based on the observation that levels of intact IGFBP-3 were
decreased, and IGFBP-3 protease activity increased, in newly diagnosed
insulin-dependent diabetes mellitus patients with full restoration
after insulin treatment. A possible role for GH in the regulation of
IGFBP-3 proteolysis was suggested by a study of Lassarre et
al. (17) because the relative amount of proteolysed IGFBP-3 on WIB
was increased in GHD patients and decreased in untreated acromegaly.
Those findings are not supported by our data. The reason for this
discrepancy is obscure but may be caused by differences in the
antibodies used. The antibody used by Lassarre et al., in
contrast to the antibody used in the present study, has a decreased
affinity for the 30-kDa fragments, which would tend towards an
underestimation of the 30-kDa fragments, when compared with the intact
IGFBP-3. This tendency, which might become even more pronounced in sera
with high concentrations of intact IGFBP-3 (acromegaly), compared with
conditions with a low concentration of intact IGFBP-3 (GHD). In
addition to WIB, we also assessed the IGFBP-3 protease activity by an
in vitro protease assay, a method that is independent of
antibody detection, and we found results similar to those obtained by
WIB. The IRMA used for measurement of IGFBP-3 in the present study has
been reported to detect intact IGFBP-3, as well as fragments of
IGFBP-3 (9, 26). WLB, on the other hand, only detects intact
IGFBP-3; and it is characteristic that sera with a low proteolytic
activity show a close relationship between immunoassayable
IGFBP-3 and the IGFBP-3 doublet in WLB, whereas sera with a high
proteolytic activity, show discrepancy between immunoassay and WLB
(12). Thus, the very similar pattern of WLB and IRMA IGFBP-3 in our
material (Fig. 3) further supports the view that GH has no major impact
on IGFBP-3 protease activity.
Fielder et al. (19) examined the effect of hypophysectomy on IGFBP-3 proteolysis in virgin and pregnant rats, and they found no effect of hypophysectomy in either case. In contrast, Rutishauser et al. (18) found that serum from normal, nonpregnant rats contains a proteolytic activity against IGFBP-3 that disappears after hypophysectomy. Hypophysectomy is a radical intervention that leads not only to GH/IGF-I deficiency but also to a deficiency in corticoids, sex steroids, and thyroid hormones, all factors whose influence on IGFBP-3 proteolysis is unknown. Despite this, it was reported in that study that the proteolytic activity reappeared after GH administration. This suggests that the protease is specifically dependent on GH, but by a different mechanism than through the induction of IGF-I, because administration of IGF-I had no effect on the IGFBP-3 proteolytic activity in the hypophysectomized rats. Patients with Laron-type dwarfism are characterized by absolute GH insensitivity and extremely low levels of IGF-I (27, 28). An increased IGFBP-3 proteolytic activity has been suggested to be present in serum from these patients (7, 29). However, in a clinical trial of IGF-I administration, IGFBP-3 proteolysis also was examined and did not differ from that in healthy, nonpregnant subjects; and no effect of IGF-I administration was observed (30).
The fact that in vitro IGFBP-3 protease in the present study was significantly increased in acromegalic patients, compared with GHD patients, and that in vivo IGFBP-3 protease was significantly increased in healthy subjects after GH administration, does not support the view that the IGFBP-3 protease is suppressed by GH. Most of the conditions in which IGFBP-3 proteolysis has been shown are characterized by insulin resistance (10) or insulinopenia (9). Because hypersecretion and exogenous administration of GH are known to cause insulin resistance, the observed apparent effects of GH on IGFBP-3 proteolysis may actually be caused by changes in insulin sensitivity. However, to investigate this, clamp studies should be performed to relate the IGFBP-3 protease to insulin sensitivity. Also, the possible effect of age on IGFBP-3 proteolysis has never been investigated. In the present study, the healthy subjects were considerably younger than the two other groups that were studied, and it cannot be excluded from present knowledge that this has influenced our results. For the same reasons, it should be emphasized that our study relates only to patients with GHD of adult onset, and no conclusions can be made from this study on IGFBP-3 proteolysis in pediatric GHD, a condition that may differ significantly in several ways.
In conclusion, we determined IGFBP-3 proteolysis in sera from subjects with different GH status and the individual changes in IGFBP-3 proteolysis in response to changes in GH status by two independent methods. We conclude that GH status has no major impact on serum IGFBP-3 proteolytic activity in humans.
Acknowledgments
We are indebted to Karen Mathiassen, Kirsten Nyborg, and Ninna Rosenqvist for their skilled technical assistance.
Footnotes
1 This work was supported by the the Danish Diabetes Association; the
Novo Foundation; the Nordic Insulin Foundation; the Aage Louis Petersen
Foundation; the Institute of Experimental Clinical Research, University
of Aarhus; and the Danish Medical Research Council, Grant no. 9600822
(Aarhus University-Novo Nordisk Centre for Research in Growth and
Regeneration) and Grant no. 9700592 (to A.F.). 
Received September 4, 1997.
Revised December 5, 1997.
Accepted December 17, 1997.
References
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