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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 10 3534-3541
Copyright © 1998 by The Endocrine Society

Original Studies

Short Stature Associated with High Circulating Insulin-Like Growth Factor (IGF)-Binding Protein-1 and Low Circulating IGF-II: Effect of Growth Hormone Therapy1

A. Barreca, M. Bozzola, A. Cesarone, P. H. Steenbergh, P. E. Holthuizen, F. Severi, G. Giordano and F. Minuto

Department of Endocrinology and Metabolism, University of Genova (A.B., A.C., G.G., F.M.), 16132 Genova; and the Department of Pediatrics, University of Pavia (M.B., F.S.), 27100 Pavia, Italy; and the Laboratory for Physiological Chemistry, Utrecht University (P.H.S., P.E.H.), 3508 TA Utrecht, The Netherlands

Address all correspondence and requests for reprints to: Dr. A. Barreca, Department of Endocrinology and Metabolism, University of Genova, 16132 Genova, Italy.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We report a case of short stature associated with high circulating levels of insulin-like growth factor (IGF)-binding protein-1 (IGFBP-10 and low levels of IGF-II responsive to pharmacological treatment with GH. Our patient suffered severe growth failure from birth (2.06 SD below the mean for normal full-term boys, and 5.2 and 7.3 SD below the mean at 5 and 10 months). Studies carried out before referral to our pediatric unit included normal 46,XY karyotype and normal encephalic imaging. Other endocrine and metabolic alterations and other systemic diseases were excluded. At 1.7 yr of age (length, 6.1 SD; weight, 4.6 SD; head circumference, 1.4 SD below the mean, respectively) the patient was referred to our pediatric unit. The baseline GH concentration was 31 µg/L, and the peak after an arginine load was 59.6 µg/L. In the same samples GH bioactivity was nearly superimposable (RIA/Nb2 bioactivity ratio = 0.9). Fasting insulin and glucose concentrations were 7.4 µU/mL and 65 mg/dL, respectively, both normally responsive to an oral glucose load. GH insensitivity was excluded by a basal IGF-I concentration (64 ng/mL) in the normal range for 0- to 5-yr-old boys and its increase after 2 IU/day hGH administration for 4 days. IGFBP-3 (0.5 µg/mL) was slightly reduced, whereas IGFBP-1 (2218 and 1515 ng/mL in two different basal samples) was well above the normal values for age and was suppressible by GH (maximum suppression, -77% at 84 h) and glucose load (maximum suppression, -46% at 150 min). The basal IGF-II concentration was below the normal range (86 ng/mL), whereas IGFBP-2 was normal (258 ng/mL). Analysis of the promoter region of IGFBP-1 and IGF-II failed to find major alterations. Neutral gel filtration of serum showed that almost all IGF-I activity was in the 35- to 45-kDa complex, coincident with IGFBP-1 peak, while the 150-kDa complex was absent, although the acid-labile subunit was normally represented.

At 2.86 yr (height, 65.8 cm; height SD score, -7.3; height velocity SD score, -5) the patient underwent treatment with 7 IU/week human GH; after 4 months, the patient’s height was 68.5 cm (height SD score, -6.9) corresponding to a growth ve locity of 8.3 cm/yr (0.3 height velocity SD score). IGFBP-1 was reduced (216 ng/mL), although still in the high range, whereas IGF-I (71 ng/mL), IGFBP-3 (0.62 µg/mL), and IGF-II (111 ng/mL) were only slightly increased. The IGF-I profile showed activity in the 150-kDa region.

In conclusion, we speculate that the increased IGFBP-1 values found in this patient produce 1) inhibition of IGF-I biological activity and, therefore, a resistance to IGF-I not due to a receptor defect for this hormone; 2) inhibition of formation of the circulating 150-kDa ternary complex and, therefore, an accelerated clearance rate of IGF peptides; 3) inhibition of the feedback action on GH, leading to increased GH levels, which could suggest the diagnosis of GH insensitivity syndrome; and 4) inhibition of body growth.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
SHORT stature secondary to a pathology involving the GH-insulin-like growth factor I (IGF-I) axis is primarily caused by GH deficiency due to pituitary disease or hypothalamic dysfunction. In this case, circulating GH and IGF-I concentrations are both abnormally low. Other causes involve GH insensitivity, the most common being Laron-type dwarfism, caused by a complete or partial GH receptor defect that makes GH therapy unsuccessful (1). Another cause of GH insensitivity has recently been described (2) due to a single missense mutation in the GH gene; this generates a GH molecule that not only cannot activate the receptor but also inhibits the action of exogenous natural GH because of its greater affinity for GH-binding protein and GH receptors. On the other hand, short stature due to biologically inactive GH, first described by Kowarski et al. (3), responds appropriately to treatment with exogenous GH. All of these forms of short stature are characterized by high GH and low IGF-I circulating concentrations. However, insensitivity to IGF-I, possibly related to defective IGF receptors and characterized by normal GH and elevated IGF-I concentrations, has also been described (4). Finally, growth failure associated with deletion of the IGF-I gene has very recently been described (5).

A particular aspect of the physiology of the IGFs regards the functions of the specific binding proteins (IGFBPs) with which they circulate under the form of high mol wt complexes (6). It has been demonstrated that IGFBPs also exert a modulatory role on IGF bioactivity. Indeed, by using different IGFBPs in several cell systems, both an inhibitory and a stimulatory effect have been shown (7). In particular, the inhibitory actions of IGFBP-1 have been shown by various in vitro studies (8, 9, 10) as well as by administration of recombinant IGFBP-1 to animals and by IGFBP-1 transgenic models (11, 12, 13).

We report a case of short stature associated with high circulating levels of IGFBP-1 and low levels of IGF-II responsive to pharmacological treatment with GH.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Case report

After an uncomplicated pregnancy, our patient was delivered at term by cesarean section for cephalo-pelvic disproportion. His birth length was 46 cm (2.06 SD below the mean for normal full-term boys), but his weight was 3350 g (0.28 SD above the mean); his head circumference was 35 cm (0.5 SD below the mean). His perinatal course was normal except for transient hypoglycemia. His father’s height was 170 cm (0.8 SD below the mean), whereas his mother measured only 144.3 cm (3 SD below the mean), respectively.

Our patient suffered severe growth failure from birth (reported as 5.2 and 7.3 SD below the mean at 5 and 10 months, respectively). Studies carried out before referral to our pediatric unit included normal 46,XY karyotype, a basal serum GH of 28.4 µg/L, and a peak serum GH concentration of 67.1 µg/L after arginine administration. His basal IGF-I concentration was within normal limits for age (48 ng/mL at 1.4 yr), and no response was reported in response to exogenous human (h) GH (0.1 IU/kg, im, for 4 days). Computed tomography revealed normal encephalic imaging. Other endocrine and metabolic alterations and other systemic diseases were excluded by appropriate diagnostic procedures performed before and after referral to our pediatric unit. In particular, malnutrition and liver disease were excluded.

At 1.7 yr of age the patient was referred to our pediatric unit with a possible diagnosis of GH insensitivity syndrome. The boy’s length was 64.7 cm (6.1 SD below the mean), weight was 7100 g (4.6 SD below the mean), and head circumference was 46.5 cm (1.4 SD below the mean). At 2.5 yr (height, 65.5 cm; weight, 7100 g; head circumference, 46.5 cm), bone age, determined according to the method of Greulich and Pyle (14), was 1 yr.

Endocrine studies

GH secretion was evaluated by arginine stimulation test using 0.5 g/kg BW arginine iv in 15 min and sampling for GH every 30 min for 120 min. IGF-I generation test was performed by administering hGH (2 IU/d) for 4 days and sampling 12, 60, and 84 h after the first administration. An oral glucose load was administered as 1.75 g/kg BW glucose, and blood glucose, insulin, and IGFBP-1 were evaluated 0, 60, 120, and 150 min after the start of administration.

Hormone assays

IGF-I was measured by double antibody RIA using immunochemicals and tracer provided by Medgenix (Fleurus, Belgium). The sensitivity of the assay was 150 pg/mL; the intra- and interassay coefficients of variation were 6% and 7.5%, respectively. The normal range for 0- to 5-yr-old subjects is 18–274 ng/mL for IGF-I. IGF-II was measured by double antibody RIA using a monoclonal antibody provided by Sera-Lab (Techno-genetics, Trezzano, Italy) and [125I]IGF-II provided by Amersham (Aylesbury, UK). The standard curve was performed using recombinant IGF-II. The sensitivity of the assay was 90 pg/mL; the intra- and interassay coefficients of variation were 6% and 9%, respectively. The IGF-II concentration in a group of 10 age-matched control boys ranged from 298–428 ng/mL. No cross-reactivity was found between IGF-I and IGF-II with the respective antibodies used in the assays up to concentrations of 500 ng/mL of both peptides. To avoid interference from binding proteins, single plasma ethylenediamine tetraacetate samples were treated with acid-ethanol, according to the method of Daughaday et al. (15).

IGFBP-1 was measured by immunoradiometric assay using reagents and tracer provided by Diagnostic Systems Laboratories, Inc. (Webster, TX). The sensitivity of the assay was 125 pg/mL; the intra- and interassay coefficients of variation were 2.5% and 4.6%, respectively. The normal range for age-matched subjects is 10–120 ng/mL. IGFBP-2 levels were determined by double antibody RIA using a nonequilibrium technique as described by Clemmons et al. (16). Specific IGFBP-2 antiserum was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and the standard was a pure IGFBP-2 preparation obtained by DNA recombinant technology (ImmunoKontact, Frankfurt, Germany). IGFBP-3 was measured by immunoassay using reagents and tracer provided by Bioclone Australia (Narrickville, Australia). The sensitivity of the assay was 3.5 ng/mL; the intra- and interassay coefficients of variation were 4.25% and 6.6%, respectively. The normal range for age-matched subjects is 0.9–3.3 µg/mL.

Serum GH concentrations were measured by both RIA and bioassay (Nb2 BA). GH was measured by RIA using a commercial kit (Sorin Diagnostics, Saluggia, Italy). The Nb2 BA was carried out in the pediatric endocrinology section at the Universitats-Kinderklinik (Tubingen, Germany) as previously described (17).

Interleukin-6 (IL-6) was measured by enzyme-linked immunosorbent assay, which uses a specific antibody for hIL-6 (Endogen, Inc., Woburn, MA). According to the manufacturer, the sensitivity of this assay is less than 1 pg/mL. All samples were tested in the same assay. The intraassay coefficient of variation was 7.2%.

Serum chromatographic profiles

One milliliter of plasma obtained from the patient before and after GH administration and 1 mL of a pool of sera from 10 age-matched controls were gel filtrated by fast protein liquid chromatography on HiPrep 16/60 S-200 column equilibrated in phosphate-buffered saline, pH 7.2. Samples were eluted at 0.8 mL/min, and fractions were collected at 0.8-min intervals. Immunoreactive IGF-I and IGFBP-1 were evaluated in fractions pooled at 0.05 Kav intervals. In these conditions the 150-kDa ternary complex elutes predominantly at 0.10–0.15 Kav, whereas the 35–45 binary complexes elute predominantly at 0.25–0.30 Kav.

Immunoblot analysis of IGFBP-1, IGFBP-3, and the acid-labile subunit (ALS) of the 150K complex

Characterization of IGFBP-1 and IGFBP-3 was performed on serum samples (3 and 6 µL for IGFBP-1 and 5 µL for IGFBP-3), whereas the ALS was evaluated in both serum samples (5 µL) and the fractions eluted at Kav 0.05–0.35 (>44 kDa) obtained after gel filtration of plasma before hGH administration (10 µL serum equivalent). Samples were denatured and fractionated under nonreducing conditions on 10% (for ALS) or 12.5% (for IGFBP-3) SDS-PAGE, then transferred electrophoretically to Hybond C-extra nitrocellulose membranes (Amersham). After transfer, nonspecific binding sites were blocked by immersing the membranes in Tris-buffered saline-Tween (TBS-T: 0.02 mol/L Tris base, 0.137 mol/L NaCl, and 0.5% Tween-20, pH 7.6, with 1 mol/L HCl) containing 5% nonfat dry milk (Bio-Rad, Hercules, CA) for 1 h at 22 C on an orbital shaker, washed five times with TBS-T, and then incubated for 16 h at 4 C with a 1:2000 dilution of anti-IGFBP-1 antiserum (Upstate Biotechnology), a 1:5000 dilution anti-IGFBP-3 antiserum (provided by Celtrix Pharmaceuticals, Inc., Santa Clara, CA), or a 1:5000 dilution antiserum to a synthetic N-terminal fragment of human ALS (provided by Dr. P. D. K. Lee, Diagnostics Systems Laboratories) in TBS-T containing 1% BSA. After incubation, the membranes were washed five times with TBS-T and incubated for 1 h at 22 C with a 1:3000 dilution of horseradish peroxidase-linked antirabbit Ig (enhanced chemiluminescence; ECL, Amersham, Aylesbury, UK), washed as before, and immersed for 1 min in the enhance chemiluminescence detection solution. Subsequently, membranes were exposed for about 0.5 min to generate immunoblots.

Genomic DNA isolation and PCR protocol

Chromosomal DNA was extracted from peripheral blood lymphocytes obtained from the patient, his parents, and unrelated control individuals as previously described (18).

Amplification of specific regions of the IGFBP-1 promoter and of the IGF-II promoter P1 was performed by PCR, using native Pfu polymerase (Stratagene, La Jolla, CA) for 30 cycles under standard conditions. Products were analyzed on 1% agarose gel. Two PCR products, corresponding to the IGF-II promoter P1, positions -750 to -381 and -384 to -6, were cloned into the pBSKS+ vector (Stratagene). Double stranded sequencing of the inserts was performed using the Pharmacia Biotech (Uppsala, Sweden) T7 sequencing kit.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Endocrine studies

The baseline GH concentration was 31 µg/L, and the peak after an arginine load was 59.6 µg/L. In the same samples GH bioactivity was nearly superimposable (RIA/Nb2 BA ratio = 0.9; Fig. 1Go).



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  		Figure 1. Immunoreactive and bioactive serum GH levels in the patient after an arginine load.

 
Fasting insulin and glucose concentrations were 7.4 µU/mL and 65 mg/dL, respectively, on one occasion. The oral glucose load excluded the presence of a form of insulin resistance (glucose, 65, 164, 35, and 47 mg/dL; insulin, 1, 8, 4, and 5.8 µU/mL at 0, 60, 120, and 150 min). Serum cortisol was 10.9 µg/dL (normal, 7–25).

The basal IGF-I concentration (64 ng/mL) was in the normal range for 0- to 5-yr-old boys and increased appropriately after administration of 2 IU/day hGH (Fig. 2Go). IGFBP-3 (0.5 µg/mL) was slightly reduced and increased slightly after GH (Fig. 2Go). The IGFBP-1 concentration (2218 and 1515 ng/mL in two different basal samples) was well above the normal values for age and was suppressible by GH [maximum change ({Delta}), -77% at 84 h; Fig. 2Go] and glucose load ({Delta}, -46 at 120 min).



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  		Figure 2. IGF-I, IGFBP-3, and IGFBP-1 circulating concentrations in the patient before and during the administration of 2 IU/day of GH for 4 days. Normal ranges for age-matched subjects are 18–274 ng/mL for IGF-I, 0.9–3.3 µg/mL for IGFBP-3, and 10–120 ng/mL for IGFBP-1.

 
Serial dilution of two patients’ serum samples showed parallelism with the standard curve in the RIA for IGFBP-1 (Fig. 3Go). By specific immunoblot (using another antibody), IGFBP-1 appeared to be present in greater amounts than in a pool of age-matched controls and had a normal molecular mass of 28–31 kDa (Fig. 4Go).



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  		Figure 3. Competitive binding curve on IGFBP-1 antiserum of two different basal serum samples from the patient, showing a displacement curve parallel to the standard used in the immunoassay and excluding any interference from unknown substances possibly contained in the patient’s serum.

 


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  		Figure 4. Immunoblot analysis of IGFBP-1 performed on serum samples (3 and 6 µL) obtained from the patient under basal conditions (lane 1), after 4 days administration of 2 IU/day GH (lane 2), after 4 months administration of 1 IU/day of GH (lane 3), and from a pool of sera from 10 age-matched controls (lane 4).

 
The chromatographic profile of IGF-I and IGFBP-1 after gel filtration in neutral conditions revealed that almost all IGF-I activity, basally as well as after the GH stimulation test, was in the 35- to 45-kDa complex, coincident with the IGFBP-1 peak, whereas the 150-kDa complex visible in normal serum (Figs. 5DGo and 6DGo) was absent (Fig. 5Go, A and B, and 6, A and B). The latter was seen after 4 months of treatment with hGH, when IGFBP-1 was drastically reduced (Figs. 5CGo and 6CGo).



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  		Figure 5. Elution profiles of immunoreactive IGF-I obtained by neutral gel filtration of 1 mL plasma from the patient under basal conditions (A), after 4-day administration of 2 IU/day GH (B), after 4-month administration of 1 IU/day GH (C), and from a pool of sera from 10 age-matched controls (D).

 


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  		Figure 6. Elution profiles of immunoreactive IGFBP-1 obtained by neutral gel filtration of 1 mL plasma from the patient under basal conditions (A), after 4-day administration of 2 IU/day GH (B), and after 4-month administration of 1 IU/day GH (C) and from a pool of sera from 10 age-matched controls (D).

 
The immunoblot demonstration of the presence of ALS in the basal serum sample in the appropriate mol wt range after neutral gel filtration (Fig. 7AGo) and in an amount comparable to that of normal age-matched subjects (Fig. 7BGo) excludes the possibility that the absence of the ternary IGF-I complex is due to the absence of this subunit. Furthermore, no difference between normal and patients’ sera was evidenced by immunoblot of IGFBP-3 proteolytic fragments (Fig. 7CGo).



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  		Figure 7. A, Immunoblot analysis of ALS performed on fractions II–VII eluted at Kav 0.05–0.35 after gel filtration of 1 mL plasma obtained from the patient under basal conditions (corresponding to the fractions shown in A of Figs. 5Go and 6Go). B, Immunoblot analysis of ALS performed on serum samples obtained from the patient under basal conditions (P) and from a pool of sera from 10 age-matched controls (C). C, Immunoblot analysis of IGFBP-3 performed on serum samples obtained from the patient under basal conditions (P) and from a pool of sera from 10 age-matched controls (C). The immunoblots were performed as detailed in Materials and Methods.

 
The basal IGF-II concentration was below the normal range (86 ng/mL), whereas IGFBP-2 and IL-6 were normal (258 ng/mL and 3.16 pg/mL).

The following clinical and laboratory data of the parents are relevant: height (SD), 0.8 and -3; IGF-I, 139 and 237 ng/mL; IGF-II, 524 and 416 ng/mL; IGFBP-1, 6.1 and 3.3 ng/mL; and IGFBP-3, 3.5 and 3.8 µg/mL for the father and mother, respectively.

Molecular studies

Using purified chromosomal DNA as template, two specific fragments of the promoter region of the IGFBP-1 gene (19) were amplified by PCR, together spanning the proximal promoter region from -720 to +37. No differences in size or quantity were detected between the amplified fragments using chromosomal DNA from the patient, both parents, and an unrelated control individual.

The low serum IGF-II concentrations suggested a possible defect in the postnatally active promoter P1 of the IGF-II gene. To test this hypothesis, two fragments of P1 of the IGF-II gene, located between positions -811 to -333 and -404 to +37 (20), were amplified by PCR and compared. Again, identical results for the patient, parents, and the unrelated control were obtained. To test the integrity of P1 at the nucleotide level and to check for possible mutations in transcription factor-binding sites, the two P1-derived PCR fragments from all four individuals were subcloned after appropriate restriction enzyme digestion and were subjected to nucleotide sequence analysis. No differences in the patient’s primary sequence of P1 between positions -750 and -6 were detected that might explain the low levels of circulating IGF-II in the patient.

Effect of hGH treatment

At 2.86 yr (height, 65.8 cm; height SD score, -7.3; height velocity SD score, -5) the patient underwent treatment with 7 IU/week hGH; after 4 months, the patient’s height was 68.5 cm (height SD score, -6.9) corresponding to a growth velocity of 8.3 cm/yr (0.3 height velocity SD score). IGFBP-1 was reduced (216 ng/mL), although still in the high range, whereas IGF-I (71 ng/mL), IGFBP-3 (0.62 µg/mL), and IGF-II (111 ng/mL) were only slightly increased. One month after discontinuation of therapy, IGFBP-1 rose again to 944 ng/mL.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
An inverse correlation between fetal size and maternal serum or fetal blood IGFBP-1 concentrations has been widely shown (21, 22, 23, 24). In postnatal life, a syndrome of resistance to IGF-I has been described in which in vitro overproduction of a 32,000-kDa binding protein by skin fibroblasts was demonstrated (25). Ours is the first description of a case of postnatal growth failure associated with circulating IGFBP-1 excess. Indeed, until now, growth failure has been ascribed to a defect in GH secretion or GH receptor availability and function, to primary or secondary IGF-I deficiency, or to an IGF-I receptor deficit (1, 2, 3, 4, 5). A number of criteria have been proposed to evaluate the primary role of some component of the GH-IGF axis as a causative factor in a child with growth retardation. In addition to auxological criteria, it is necessary to progressively evaluate serum IGF-I and/or IGFBP-3 concentrations, the IGF-I/IGFBP-2 ratio, spontaneous or stimulated GH secretion, and GH-binding protein (26). Although GH exerts an inhibitory effect on the synthesis of IGFBP-1 as well as IGFBP-2 (16, 27, 28, 29, 30, 31, 32, 33, 34, 35), IGFBP-1 is not usually considered, because, unlike IGFBP-3 and IGFBP-2, it shows marked diurnal variations due to changes in metabolic status (36). Moreover, owing to its short half-life and lower circulating levels, IGFBP-1 does not usually have an important role in stabilizing circulating IGF (6).

In this patient, the slightly increased basal GH concentration and robust response to stimulation in the absence of malnutrition prompted us to exclude a form of GH resistance as a cause of growth retardation. GH insensitivity was excluded by the fact that the basal IGF-I concentration was in the normal range for a 3-yr-old boy and increased appropriately during the administration of 2 IU hGH/day for 4 days. Biologically inactive GH was excluded by Nb2 BA performed on the samples obtained in basal conditions and after an arginine load. We therefore decided to look into other parameters of the IGF system.

The patient showed slightly reduced circulating IGFBP-3 and normal IGFBP-2, but low IGF-II and greatly increased IGFBP-1 concentrations. Basal IGFBP-1 levels were even higher than those found in patients affected with GH insensitivity, in whom fasting IGFBP-1 has been reported to be always less than 300 ng/mL (37, 38). The parallelism between our patient’ serum and the IGFBP-1 standard rendered it unlikely that some unknown substance in the patient’s serum gave artifactually high values with the antibody used. Moreover, a specific immunoblot using a different antibody confirmed that IGFBP-1 was present in greater amounts than in a pool of age-matched control sera and had a normal molecular mass. Neutral gel filtration demonstrated that almost all circulating IGF-I activity was in the 35- to 45-kDa complex, coincident with the IGFBP-1 peak, thus showing that this protein is able to bind the IGF peptides. Although the acid-labile subunit and IGFBP-3 were present, the molar concentration of IGFBP-1, almost an order of magnitude higher than that of IGFBP-3, prevented, despite its lower affinity for IGF (39), the binding of the IGF to IGFBP-3 and, therefore, the formation of the ternary complex with ALS (40, 41). This is confirmed by the finding that only after 4 months of treatment with hGH, when IGFBP-1 was reduced by an order of magnitude, was the IGF-I activity shifted in the 150-kDa region, although at that time there was only a slight increase in the levels of IGF-I and IGFBP-3.

Given that the IGFBP-1 protein is synthesized in a biologically active form, we assumed that the coding region was intact. Therefore, we analyzed the promoter region of IGFBP-1, but found no insertions and/or deletions; however, for a complete analysis, the region should be sequenced. The fact that no aberrations in size or quantity were detected implies that two intact copies of the IGFBP-1 gene are present in the patient.

In agreement with the molecular studies, we were unable to identify any obvious physiological factor responsible for IGFBP-1 dysregulation. Indeed, glucose concentrations and fasting insulin levels, which are the primary regulators of IGFBP-1 (36), were in the normal range and normally responsive to an oral glucose load, which also induced the expected reduction in IGFBP-1. IL-6, a cytokine whose stimulatory effect on IGFBP-1 synthesis prevails over the inhibitory action of insulin (42), was normal. As the inhibitory action of IGFs on IGFBP-1 synthesis is equal to or greater than that of insulin (36, 43, 44, 45), the low IGF-II concentration present in the patient could be the cause of the high IGFBP-1 basal values. Nevertheless, chronic treatment with hGH resulted in a marked decrease in circulating IGFBP-1, with only a slight increase in IGF-I and IGF-II. At this time, we cannot exclude that the excess circulating IGFBP-1 could be due to unidentified substances that stimulate IGFBP-1 expression, possibly by stimulating cAMP (46, 47, 48) or inhibiting protein kinase C (49).

Regarding the significantly low IGF-II concentration in the patient’s serum, PCR analysis of the gene showed that promoter P1 of IGF-II located between insulin and the first exon of IGF-II is intact. As IGF-II expression after birth is regulated by the P1 promoter of the IGF-II gene (50), the sequence of the proximal promoter P1 region was determined and checked for putative mutations in the regulatory elements of this promoter. A region of 700 nucleotides showed no aberrations. This region includes the established regulatory elements, such as an essential Sp1 site and a C/EBP{alpha} binding site, which are both required for normal activity of P1 (51). At present, we cannot exclude that the low circulating levels of IGF-II are due to an excess of IGF-II/mannose-6-phosphate receptor, which has been shown to cause internalization and degradation of IGF-II (52, 53).

The finding that the mother has a short stature (3 SD below the mean in normal subjects) without any alteration of circulating IGFs and IGFBPs is puzzling. A priori we assumed that a common genetic factor was responsible for the short stature in both mother and son. However, the absence of elevated circulating IGFBP-1 and low circulating IGF-II in the mother seems to exclude a common alteration in the IGF-IGFBP system.

Contrasting data are reported in the literature concerning the effect of IGFBP-1 excess on carbohydrate metabolism in transgenic mice (12, 54) as well as in animals injected with IGFBPs (6, 55). With regard to transgenic mice, a possible explanation resides in the different organs in which IGFBP-1 is overexpressed. We did not observe alterations of carbohydrate metabolism in our patient. The mechanism by which IGFBP-1 increases blood glucose has been postulated to be the inhibitory effect of IGFBP-1 on the peripheral insulin-like effects of IGFs. This could have been counteracted in our patient by the shift of IGF peptides in the circulation from the ternary to the binary complex, which has been postulated to increase the insulin-like effects of these peptides.

The decrease in IGFBP-1 after hGH administration is in agreement with the finding that in vitro GH is able to inhibit IGFBP-1 synthesis in rats (27, 28, 29). The finding that in this patient, GH administration induces a decrease in serum IGFBP-1 concentrations is consistent with the evidence that GH in humans exerts a suppressive effect on IGFBP-1 only at pharmacological concentrations (56). This is similar to the findings in Turner’s syndrome (57) and in chronic renal failure (58), conditions associated with normal or elevated endogenous serum GH levels. Indeed, hGH treatment for 4 months reduced the circulating IGFBP-1 and, concomitantly, improved the patient’s height velocity, while discontinuation of the therapy increased IGFBP-1 again.

The growth stimulation in response to exogenous hGH may have been to some degree secondary to the reduction of IGFBP-1. Such an effect would be in agreement with in vivo and in vitro studies establishing a role for IGFBP-1 in inhibiting IGF-stimulated growth and differentiative function (36). Moreover, overexpression of IGFBP-1 in transgenic mice resulted in impaired somatic growth and hyperglycemia (12). As IGF-I bound in the 150K complex seems to represent the most potent anabolic form of circulating IGF-I, reduced formation of the ternary complex may also contribute to the decreased IGF-I growth-promoting activity in our patient (7).

In conclusion, we speculate that the increased IGFBP-1 values found in this patient produce 1) inhibition of IGF-I biological activity and, therefore, a resistance to IGF-I not due to a receptor defect for this hormone; 2) inhibition of the formation of the circulating 150-kDa ternary complex and, therefore, an accelerated clearance rate of IGF peptides; 3) inhibition of the feedback action on GH, leading to increased GH levels, which could suggest the diagnosis of the GH insensitivity syndrome; and 4) inhibition of body growth.

We expect that following this case report, IGFBP-1 will be considered in the course of diagnosis of growth failure unexplained by a defect in any of the other components of the GH-IGF-IGFBP system known to cause it. This will identify new cases and, hopefully, discover the cause of the IGFBP-1 excess.


   Acknowledgments
 
The authors are indebted to Dr. M. W. Elminger (Tubingen, Germany) for performance of Nb2 cell assay, to Dr. P. D. K. Lee (Diagnostics Systems Laboratories, Inc., Webster, TX) for providing the antiserum to a synthetic N-terminal fragment of human ALS, to Celtrix Pharmaceuticals, Inc. (Santa Clara, CA), for providing the antiserum to IGFBP-3, and to Dr. Judson J. Van Wyk (University of North Carolina, Chapel Hill, NC) for critical review of the manuscript.


   Footnotes
 
1 This work was supported by research grants from Ministero dell’Università e della Ricerca Scientifica (40% and 60%). Back

Received April 10, 1998.

Revised June 29, 1998.

Accepted July 13, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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