Consensus statement on the diagnosis and treatment of children with idiopathic short stature: a summary of the Growth Hormone Research Society, the Lawson Wilkins Pediatric Endocrine Society, and the European Society for Paediatric Endocrinology Workshop.

OBJECTIVE: Our objective was to summarize important advances in the management of children with idiopathic short stature (ISS). PARTICIPANTS: Participants were 32 invited leaders in the field. EVIDENCE: Evidence was obtained by extensive literature review and from clinical experience. CONSENSUS: Participants reviewed discussion summaries, voted, and reached a majority decision on each document section. CONCLUSIONS: ISS is defined auxologically by a height below -2 sd score (SDS) without findings of disease as evident by a complete evaluation by a pediatric endocrinologist including stimulated GH levels. Magnetic resonance imaging is not necessary in patients with ISS. ISS may be a risk factor for psychosocial problems, but true psychopathology is rare. In the United States and seven other countries, the regulatory authorities approved GH treatment (at doses up to 53 microg/kg.d) for children shorter than -2.25 SDS, whereas in other countries, lower cutoffs are proposed. Aromatase inhibition increases predicted adult height in males with ISS, but adult-height data are not available. Psychological counseling is worthwhile to consider instead of or as an adjunct to hormone treatment. The predicted height may be inaccurate and is not an absolute criterion for GH treatment decisions. The shorter the child, the more consideration should be given to GH. Successful first-year response to GH treatment includes an increase in height SDS of more than 0.3-0.5. The mean increase in adult height in children with ISS attributable to GH therapy (average duration of 4-7 yr) is 3.5-7.5 cm. Responses are highly variable. IGF-I levels may be helpful in assessing compliance and GH sensitivity; levels that are consistently elevated (>2.5 SDS) should prompt consideration of GH dose reduction. GH therapy for children with ISS has a similar safety profile to other GH indications.

Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity.

Insulin and insulin-like growth factors (IGFs) mediate a variety of signals involved in mammalian development and metabolism. To study the metabolic consequences of IGF-I deficiency, we used the liver IGF-I-deficient (LID) mouse model. The LID mice show a marked reduction (approximately 75%) in circulating IGF-I and elevated growth hormone (GH) levels. Interestingly, LID mice show a fourfold increase in serum insulin levels (2.2 vs. 0.6 ng/ml in control mice) and abnormal glucose clearance after insulin injection. Fasting blood glucose levels and those after a glucose tolerance test were similar between the LID mice and their control littermates. Thus, the high levels of circulating insulin enable the LID mice to maintain normoglycemia in the presence of apparent insulin insensitivity. Insulin-induced autophosphorylation of the insulin receptor and tyrosine phosphorylation of insulin receptor substrate (IRS)-1 were absent in muscle, but were normal in liver and white adipose tissue of the LID mice. In contrast, IGF-I-induced autophosphorylation of its cognate receptor and phosphorylation of IRS-1 were normal in muscle of LID mice. Thus, the insulin insensitivity seen in the LID mice is muscle specific. Recombinant human IGF-I treatment of the LID mice caused a reduction in insulin levels and an increase in insulin sensitivity. Treatment of the LID mice with GH-releasing hormone antagonist, which reduces GH levels, also increased insulin sensitivity. These data provide evidence of the role of circulating IGF-I as an important component of overall insulin action in peripheral tissues.

Evaluation of DXA vs. MRI for body composition measures in 1-month olds.

BACKGROUND: Detailed measures of infant body composition are needed for understanding the impact of genes and environment on growth early in life. OBJECTIVE: The purpose of this study was to compare the accuracy and bias of body composition in infants. METHODS: Dual energy X-ray absorptiometry (DXA) and magnetic resonance imaging (MRI) were used to determine body composition and the trunk depot. The depots measured were total fat mass (FM), total fat-free mass (FFM) and trunk FM and FFM using DXA and MRI in 14 infants. RESULTS: None of the regression lines between DXA and MRI significantly deviate from the line of identity for any of the depots studied. However, Bland-Altman analyses revealed bias for trunk FM and trunk FFM. CONCLUSION: Our data showed DXA to be accurate (regression not significantly deviating from the line of identity), with high agreement (indicated by high R(2) ) and without bias (non-significant Bland-Altman) when estimating total FM and FFM. This could not be said for trunk estimates.

Discordance of serum and tissue somatomedin levels in growth hormone-stimulated growth in the rat.

Although the somatomedins are believed to mediate GH-induced somatic growth, circulating levels of insulin-like growth factor I (IGF-I) do not always correlate with growth rate. To evaluate a paracrine or autocrine effect that might explain this discordance, the concentrations of plasma and tissue IGF-I were compared with indices of growth in hypophysectomized rats treated with rat GH (rGH). The rats received a total of 250 micrograms rGH by either continuous pump infusion (P) or twice daily injections (I). After 4 days of treatment, the amounts of IGF-I present in acetic acid extracts of liver and kidney and in native serum were determined by RIA and related to proximal tibial epiphyseal plate width. Tibial epiphyseal plate width increased from 198 +/- 6 microns (mean +/- SE) in untreated controls to 339 +/- 13 microns in group P and 347 +/- 9 micron in group I; weight gain was 11.6 +/- 1.0 g in group P and 9.6 +/- 0.8 g in group I, while control animals lost 1.0 g. Serum concentrations of IGF-I were no different between control animals and those in the injection group (0.91 +/- 0.06 vs. 0.90 +/- 0.06 U/ml), whereas levels in the infusion group increased slightly (1.1 +/- 0.05 U/ml). In contrast, rGH administration caused tissue IGF-I to double in liver (control, 0.24 +/- 0.04 U/g; P, 0.51 +/- 0.03 U/g; I, 0.46 +/- 0.05 U/g) and nearly triple in kidney (control, 0.52 +/- 0.05 U/g; P, 1.51 +/- 0.09 U/g; I, 1.36 +/- 0.08 U/g). There was no detectable change in somatomedin-binding protein by gel exclusion chromatography. Since the rGH administered was sufficient to stimulate growth and increase tissue somatomedin levels without corresponding increases in circulating IGF-I, an autocrine or paracrine action of IGF-I appears to mediate GH's initial somatogenic actions in the young rat.

Insulin-like growth factor-I (IGF-I) enhanced proteolysis of IGF-binding protein-4 in conditioned medium from primary cultures of human decidua: independence from IGF receptor binding.

Previous studies demonstrated that human decidual cells release insulin-like growth factor-binding protein (IGFBP)-1, IGFBP-2, and a 24-kilodalton (kDa) IGFBP in culture. The accumulation of 24-kDa IGFBP, as assessed by ligand blot analysis, decreased when the cells were exposed to IGF-I, but the mechanism was not explored. In the present study, we observed that the IGF-I-mediated decrease in IGFBP-4 accumulation could be explained by increased IGFBP-4 proteolysis. Analysis by IGFBP-4 immunoblotting demonstrated a decline in 24-kDa IGFBP-4 accompanied by a marked increase in a 17- to 18.5-kDa IGFBP-4 fragment(s). In addition, when medium from IGF-I-treated cells was incubated with rat IGFBP-4, the decrease in IGFBP-4 was inhibited by chelators of divalent cations and inhibitors of serine proteases. IGF-I enhancement of IGFBP-4 proteolysis occurs independent of the type I IGF receptor. [Leu24,1-62]IGF-I, an analog with reduced receptor affinity, mimicked the effect of native IGF-I in cell culture. Additionally, alpha-IR3, a monoclonal antibody to the type I IGF receptor, did not block the effect of IGF-I. When IGF-I was incubated with medium from control cells, there was a marked decrease in 24-kDa IGFBP-4 levels and a concomitant increase in levels of a 17- to 18.5-kDa fragment(s), suggesting that IGFBP-4 complexed with IGF-I is more susceptible to proteolysis than IGFBP-4 alone. Together, these findings suggest a novel mechanism for regulation of IGF-I action in the decidua.

Mechanisms of Sertoli cell insulin-like growth factor (IGF)-binding protein-3 regulation by IGF-I and adenosine 3',5'-monophosphate.

FSH, which stimulates cAMP in the Sertoli cell, markedly lowers the concentration of insulin-like growth factor-binding protein-3 (IGFBP-3) in Sertoli cell-conditioned medium; in contrast, insulin-like growth factor-I (IGF-I) increases BP-3 expression. In this study, the mechanisms controlling the contrasting effects of cAMP and IGF-I were investigated. The abundance of BP-3 mRNA was dramatically lowered by (Bu)2cAMP, but was unaffected by IGF-I. Analyzed by ligand blot of conditioned medium, coincubation of (Bu)2cAMP and IGF-I largely eliminated the increase observed with IGF-I alone. Based on the following evidence, the effect of IGF-I appeared to be solely related to the capacity of IGF-I to interact directly with BP-3. 1) Insulin at micromolar concentrations failed to increase BP-3 abundance despite documentation by affinity cross-linking that insulin displaced [125I]IGF-I from the IGF-I receptor. 2) A synthetic IGF-I analog, [Leu24,1-62]IGF-I, which has reduced binding affinity for rat IGF-I receptor but displays high affinity for rat Sertoli cell-conditioned medium BPs, increased BP-3 abundance. 3) A synthetic IGF-I analog, B-chain mutant, which has reduced affinity for rat Sertoli cell BPs but displays normal affinity for the rat IGF-I receptor, failed to increase BP-3 abundance. 4) Human recombinant glycosylated [125I]BP-3 when added to cultured Sertoli cells was preserved in the medium when IGF-I or analogs with BP-3 affinity were present. 5) IGF-I, in dose-responsive manner, both retarded the disappearance from the medium of exogenously added human recombinant nonglycosylated BP-3 and decreased the amount of membrane-associated BP-3. These results indicate that whereas cAMP lowers BP-3 abundance in medium, most likely by markedly decreasing synthesis, IGF-I increases BP-3 accumulation by retarding its clearance by the Sertoli cell.

Glucocorticoid regulation of an insulin-like growth factor-binding protein-4 protease produced by a rat neuronal cell line.

Insulin-like growth factor-binding protein-4 (IGFBP-4) is expressed in distinct regions in the rodent brain from the perinatal period into adulthood and is postulated to modulate the action of the insulin-like growth factors (IGFs) in vivo. This study was initiated to examine the regulation of IGF-binding protein-4 (IGFBP-4) in B104 cells, a rat neuronal cell line in which IGFBP-4 is the predominant secreted IGFBP. Exposure of B104 monolayer cultures to dexamethasone reduced native IGFBP-4 abundance to less than 10% of that in control medium by 48 h. Immunoblots showed that the decline in intact 24-kilodalton IGFBP-4 was accompanied by an increase in a 16-kilodalton immunoreactive fragment. In addition, IGFBP-4 proteolytic activity in medium was increased after exposure of the cells to dexamethasone. The protease was calcium dependent and appeared to be of the serine protease class, because activity could be inhibited by phenylmethylsulfonylfluoride and aprotinin, but not antipain, leupeptin, or pepstatin. Although the proteolytically modified IGFBP-4 retained the ability to bind IGFs, the affinities were approximately 13- and 20-fold lower for IGF-I and IGF-II, respectively. These data indicate that B104 cells produce an IGFBP-4 protease that is regulated by glucocorticoids. The actions of this protease reduce the affinity of IGFBP-4 for the IGFs without abolishing binding. Because both the IGFs and glucocorticoids have important roles in brain development, it is possible that some glucocorticoid actions in the brain could be mediated by proteolysis of IGFBP-4, which, in turn, would alter IGF action.

Insulin-like growth factor-binding protein-3 (IGFBP-3) concentration in rat Sertoli cell-conditioned medium is regulated by a pathway involving association of IGFBP-3 with cell surface proteoglycans.

Rat Sertoli cells in culture produce insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 (IGFBP-3) is the predominant IGFBP. Previous studies suggested that the concentration of IGFBP-3 in Sertoli cell-conditioned medium is regulated by a clearance pathway involving association of IGFBP-3 with the Sertoli cell surface. The purpose of these studies was to characterize further the nature of the interaction of IGFBP-3 with the cell surface and to examine how this interaction might be regulating IGFBP-3 concentrations in conditioned medium. Recombinant nonglycosylated human IGFBP-3 was added to cultured Sertoli cells derived from 15-day-old rats, and the change in concentration over time in the medium was measured by [125I]IGF-I ligand blot analysis and Western blot analysis using an antiserum specific for human IGFBP-3. Chloroquine, an inhibitor of lysosomal enzymes involved in internalization of proteins and reduction of temperature, inhibited IGFBP-3 reduction in Sertoli cell medium. Soluble heparin, at a half-maximal concentration of approximately 5 microgram/ml, inhibited the decrease in IGFBP-3 in the medium. In addition, soluble heparin decreased the amount of IGFBP-3 detectable on the cell surface, as determined by performing ligand blots on plasma membrane preparations. Finally, pretreatment of Sertoli cells with sodium chlorate, an inhibitor of cell surface proteoglycan sulfation, also retarded the decrease in recombinant IGFBP-3. Taken together, these data suggest that an important mechanism influencing the concentration of IGFBP-3 3 in Sertoli cell-conditioned medium is a pathway involving IGFBP-3 interaction with the cell surface proteoglycans.

Influence of hypothyroidism on growth hormone binding by rat liver.

The binding of 125I-iodinated human GH ([125I]iodo-hGH) to crude, membrane-rich preparations from the livers of thyroidectomized male rats was examined to determine if alterations in GH binding might explain hypothyroid-induced growth failure. Membrane preparations from chronically hypothyroid rats bound nearly twice as much labeled hGH (12.8 +/- 2.5%; mean +/- 1 SD) as those from normal rats (6.6 +/- 1.4%) or thyroxine-treated thyroidectomized animals (7.0 +/- 1.1%). Binding by membrane preparations from hypothyroid rats treated with hGH for 2 weeks was 12.3%. By Scatchard analysis the apparent affinities of the membrane preparations for hGH were relatively constant (Ka = 2.30-2.88 X 10(9) M-1) among the experimental groups. Ovine PRL was only 1.4% as potent as hGH in displacing [125I]iodo-hGH from the liver preparations, indicating that hGH was bound primarily to somatogenic sites. Since administration of hGH did not reduce the binding of [125I]iodo-hGH, it is unlikely that the increase in hGH binding in hypothyroidism is mediated by a reduction in the ambient GH concentration. Furthermore, this increase in [125I]iodo-hGH binding indicates that an alteration in binding of GH to its receptor probably does not mediate either the low somatomedin levels or the growth failure that result from hypothyroidism.

Insulin-like growth factor binding protein-3 secretion from cultured rat sertoli cells: dual regulation by follicle stimulating hormone and insulin-like growth factor-I.

The insulin-like growth factors (IGFs) are found in extracellular fluids bound to carrier proteins which influence the biological activity of the IGFs. Three structurally different binding proteins (BPs) have been isolated and cloned; each has distinct tissue specific expression and unique properties. We report here that testicular cells synthesize a specific subset of these binding proteins. Ligand blot analysis and RNA blot hybridization indicates that cultured peritubular cells synthesize primarily IGFBP-2. In contrast, as determined by ligand blot, RNA blot hybridization and N-linked deglycosylated studies, IGFBP-3 is predominantly synthesized by the Sertoli cell. In a dose dependent fashion, FSH markedly reduces the levels of IGFBP-3 in Sertoli cell conditioned medium. Similarly, isoproterenol, (Bu)2cAMP and cholera toxin also markedly reduce the abundance of IGFBP-3 in conditioned media. In contrast, IGF-I increases the concentrations of IGFBP-3 with the concentration required for half-maximal stimulation, approximately 20 ng/ml. Consistent with a peritubular cell origin, IGFBP-2 may be the predominant species found in interstitial fluid. In summary, our data reveal that the IGFBPs are expressed in a cell type specific manner in the testis. The opposing effects of FSH and IGF-I on Sertoli cell IGFBP-3 expression suggests a mechanism by which the IGF-I biological activity on Sertoli cell might be influenced.

Epidermal growth factor stimulates insulin-like growth factor-binding protein-1 expression in the neonatal rat.

Insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs) appear to be important in the regulation of perinatal growth. We have shown previously that administration of epidermal growth factor (EGF) to newborn rat pups inhibits growth and decreases serum IGF-I concentrations. The experiments described here were designed to investigate the effect of EGF on the IGFBPs using ligand blots of serum and Northern analysis of hepatic RNA. EGF administration caused a rapid (within 2 h) 2-fold increase in the serum IGFBP-1 concentration. Hepatic IGFBP-1 mRNA increased even more rapidly, was increased at least 2-fold at 2 h, and remained elevated 4 h after EGF. The response to EGF was specific to IGFBP-1; IGFBP-2 hepatic mRNA content was not increased over the control value, and serum IGFBP-3 and -4 concentrations were not changed by ligand blot analysis. The IGFBP-1 response to EGF was most dramatic in the first few days of life. Although EGF lowered circulating insulin levels, EGF stimulated IGFBP-1 secretion in the presence of exogenously administered insulin. Thus, the increase in IGFBP-1 did not appear to be mediated by changes in serum insulin. These results demonstrate that EGF increases serum IGFBP-1 concentrations, probably by stimulating synthesis. The association of decreased growth and increased IGFBP-1 concentrations after EGF treatment suggests that elevated IGFBP-1 concentrations may restrict IGF bioactivity in the neonatal rat.

Insulin-like growth factor binding proteins in fetal rat mesencephalic cultures: regulation by fibroblast growth factor and insulin-like growth factor I.

In rat ventral mesencephalic cultures, IGF-I and bovine fibroblast growth factor (bFGF) act cooperatively to support the survival of dopaminergic neurons. To determine the potential role of IGFBPs in modulating the actions of IGF-I in the ventral mesencephalon, we identified the IGFBPs present in ventral mesencephalic cultures and examined their regulation by IGF-I and bFGF. In the absence of added growth factors, the major binding protein secreted from these cultures was IGFBP-2. Small amounts of IGFFBP-3 and IGFBP-4 were also detected. Addition of bFGF to the cultures increased the amounts of IGFBP-3 and IGFBP-4 released from the cells by 4.4 +/- 2.6 -fold (P < 0.1) and 11.5 +/- 3.5 -fold (P < 0.05), respectively. IGF-I, itself, had little effect on the production of IGFBPs, but when added together with bFGF increased the levels of IGFBP-3 and IGFBP-4 by 12.4 +/- 5.1 -fold (P < 0.05) and 27.4 +/- 5.3 -fold (P < 0.02), respectively. The stimulatory effect of bFGF and IGF-I on IGFBP production was apparent after a 2- to 3-day exposure of the mesencephalic cultures to the peptides. IGFBP-4, the most abundant IGFBP present in the cultures after 7 days of growth factor treatment, was immunocyto-chemically localized primarily to neurons, of which a subset were dopaminergic neurons. The addition of purified rat IGFBP-4 to the cultures in the absence of added growth factors had no effect on the survival of dopaminergic neurons, but when added with IGF-I potentiated the effect of IGF-I on neuronal survival. We propose that the up-regulation of IGFBP-4 by IGF-I and bFGF may serve to localize IGF-I to sites of action in the nervous system and thereby potentiate the neurotrophic actions of IGF-I.

Characterization of an insulin-like growth factor binding protein (IGFBP-4) produced by the B104 rat neuronal cell line: chemical and biological properties and differential synthesis by sublines.

A previous report from our laboratory described an approximately 30 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) that inhibited the binding of insulin-like growth factor I (IGF-I) by its receptor and was secreted by a subline of the B104 rat neuronal cell line. To better understand the biology of this IGFBP, it was purified from media conditioned by these B104 cells, and the chemical and biological properties of the protein were examined. The IGFBP existed as a 24 kDa form and a 28 kDa form when the conditioned media were analyzed by ligand blot. Deglycosylation studies indicated the 28 kDa species was the N-linked glycosylated form of the 24 kDa IGFBP. Multiple forms at both mol wts were found using two-dimensional electrophoresis, suggesting that there were posttranslational modifications in addition to glycosylation. The amino acid sequence of the 12 amino-terminal residues was identical to that of rat IGFBP-4. Increased synthesis of IGFBP-4 by the subline contrasted with neglible production by other B104 cells. Blot hybridization with rat IGFBP-4 complementary DNA showed differential expression of a 2.6 kilobase transcript among B104 cell lines that correlated with quantities of IGFBP-4 secreted in media. The difference persisted even when the cells were xenografted into athymic nude mice. Purified IGFBP-4 inhibited the binding of [125I]IGF-I by its receptor and blunted stimulation of [3H]thymidine incorporation by IGF-I. These findings suggest a role for IGFBP-4 in neural cell function and indicate the B104 cell lines may be a useful model for further examination of IGFBP-4 biology.

Overexpression of insulin-like growth factor-binding protein-4 (IGFBP-4) in smooth muscle cells of transgenic mice through a smooth muscle alpha-actin-IGFBP-4 fusion gene induces smooth muscle hypoplasia.

Insulin-like growth factor I (IGF-I) has been postulated to function as a smooth muscle cell (SMC) mitogen and to play a role in the pathogenesis of bladder hypertrophy, estrogen-induced uterine growth, and restenosis after arterial angioplasty. IGF-binding protein-4 (IGFBP-4) inhibits IGF-I action in vitro and is the most abundant IGFBP in the rodent arterial wall. To explore the function of this binding protein in vivo, transgenic mouse lines were developed harboring fusion genes consisting of a rat IGFBP-4 complementary DNA cloned downstream of either a -724 bp fragment of the mouse smooth muscle alpha-actin 5'-flanking region (SMP2-BP-4) or -1074 bp, 63 bp of 5'-untranslated region, and 2.5 kb of intron 1 of smooth muscle alpha-actin (SMP8-BP-4). SMP2-BP-4 mice expressed low levels of the exogenous IGFBP-4 messenger RNA (mRNA), which was not specifically targeted to SMC-rich tissue environments, and were therefore not analyzed further. Six SMP8-BP-4 transgenic lines derived from separate founders were characterized. Mating of hemizygous SMP8-BP-4 mice with controls produced about 50% transgenic offspring, with equal sex distribution. Expression of IGFBP-4 mRNA in nontransgenic littermates was maximal in liver and kidney. By contrast, transgenic IGFBP-4 mRNA expression, distinguished because of a smaller transcript size, was confined to SMC-containing tissues, with the following hierarchy: bladder > aorta > stomach = uterus. There was no transgene expression in skeletal muscle, brain, or cardiac myocytes. The abundance of IGFBP-4 measured by Western ligand blotting or by immunoblotting, was 8- to 10-fold higher in aorta and bladder of SMP8-BP-4 mice than in their nontransgenic littermates, with no change in plasma IGFBP-4 levels. Transgenic mice exhibited a significant reduction in wet weight of SMC-rich tissues, including bladder, intestine, aorta, uterus, and stomach, with no change in total body or carcass weight. In situ hybridization showed that transgene expression was targeted exclusively to the muscular layers of the arteries, veins, bladder, ureter, stomach, intestine, and uterus. Overexpression of IGFBP-4 was associated with SMC hypoplasia, a reciprocal phenotype to that of transgenic mice overexpressing IGF-I under control of the same promoter (SMP8-IGF-I). Double transgenic mice derived from mating SMP8-BP-4 with SMP8-IGF-I animals showed a modest decrease in wet weight at selected SMC tissues. Although we cannot exclude that the effects of IGFBP-4 may be IGF independent, these data suggest that IGFBP-4 is a functional antagonist of IGF-I action on SMC in vivo.

IGF-binding protein-4 expression and IGF-binding protein-4 protease activity are regulated coordinately in smooth muscle during postnatal development and after vascular injury.

Recent studies support a critical role for the paracrine IGF/IGF-binding protein system in the regulation of vascular smooth muscle cell growth. In this study we have explored the hypothesis that the abundance of individual IGF-binding proteins in smooth muscle is subject to regulation during postnatal life and in response to injury. IGF-binding protein-2 was the predominant binding protein secreted by neonatal rat vascular smooth muscle cells, whereas IGF-binding protein-4 was most prevalent in adult vascular smooth muscle cells coincident with increased IGF-binding protein-4 protease activity. After arterial injury, IGF-binding protein-4 mRNA increased, associated with greater IGF-binding protein-4 proteolytic activity, resulting in stable steady state levels of the IGF-binding protein-4 protein. Expression of pregnancy-associated plasma protein A mRNA, recently identified as an IGF-binding protein-4 protease, was expressed at higher levels in adult than neonatal vascular smooth muscle cell lines, but did not change significantly after arterial injury. The peak of immunoreactive pregnancy-associated plasma protein A from hydrophobic interaction chromatography fractions of smooth muscle cell-conditioned medium coincided, but did not fully overlap, with the fractions containing maximal IGF-binding protein-4 protease activity. In conclusion, our data point to a developmental switch from IGF-binding protein-2 to IGF-binding protein-4 in vascular smooth muscle cells postnatally. Moreover, IGF-binding protein-4 expression is coregulated with IGF-binding protein-4 protease activity, suggesting that biosynthesis and degradation of this binding protein are coordinated events important for regulating biological activity of IGF-I.

Targeted overexpression of insulin-like growth factor I to osteoblasts of transgenic mice: increased trabecular bone volume without increased osteoblast proliferation.

Insulin-like growth factor I (IGF-I) is an important growth factor for bone, yet the mechanisms that mediate its anabolic activity in the skeleton are poorly understood. To examine the effects of locally produced IGF-I in bone in vivo, we targeted expression IGF-I to osteoblasts of transgenic mice using a human osteocalcin promoter. The IGF-I transgene was expressed in bone osteoblasts in OC-IGF-I transgenic mice at high levels in the absence of any change in serum IGF-I levels, or of total body growth. Bone formation rate at the distal femur in 3-week-old OC-IGF-I transgenic mice was approximately twice that of controls. By 6 weeks, bone mineral density as measured by dual energy x-ray, and quantitative computed tomography was significantly greater in OC-IGF-I transgenic mice compared with controls. Histomorphometric measurements revealed a marked (30%) increase femoral cancellous bone volume in the OC-IGF-I transgenic mice, but no change in the total number of osteoblasts or osteoclasts. Transgenic mice also demonstrated an increase in the osteocyte lacunea occupancy, suggesting that IGF-I may extend the osteocyte life span. We conclude that IGF-I produced locally in bone osteoblasts exerts its anabolic effect primarily by increasing the activity of resident osteoblasts.

Acromegaloid patients with type A insulin resistance: parallel defects in insulin and insulin-like growth factor-I receptors and biological responses in cultured fibroblasts.

A subset of patients with the syndrome of acanthosis nigricans and insulin resistance type A is characterized by acromegaloid features in addition to hyperinsulinemia, hyperandrogenemia, and an inherent defect in insulin receptor function. It has been proposed that the acromegaloid features result from the interaction of insulin at concentrations encountered in vivo, with a functionally intact insulin-like growth factor-I (IGF-I) receptor closely related to the insulin receptor. We investigated this possibility by examining binding and hormone-stimulated [14C]glucose uptake, [3H]thymidine uptake, and receptor autophosphorylation by both insulin and IGF-I in cultured fibroblasts from two affected patients. In comparison to normal fibroblasts, [125I]insulin binding, insulin-stimulated [14C]glucose, and [3H]thymidine uptake, and insulin-stimulated autophosphorylation were each reduced by approximately 50-60% of the absolute values in controls. In contrast to expectation, each of these apparent defects in insulin binding and action were mirrored by a parallel decrease in IGF-I binding and action. Thus, [125I]IGF binding was approximately 50%, IGF-I stimulated [3H]thymidine uptake was approximately 40% and 60% of the control value, and IGF-I-stimulated receptor autophosphorylation was reduced by 40%. Incubation of fibroblasts with insulin at 25 ng/mL reduced subsequent binding of [125I]IGF-I by approximately 20% and did not enhance maximal stimulation of [3H]thymidine incorporation. We conclude that in some patients with acanthosis nigricans and acromegaloid features, IGF-I receptors of cultured fibroblasts may share the inherent defects of insulin receptor function. These in vitro data do not explain the acromegaloid features observed in vivo, suggesting that acromegaloid features are mediated by other mechanisms.

Characteristics of hepatic receptors for somatomedin-C/insulin-like growth factor I and insulin in the developing human.

Insulin and the somatomedins (Sms) are putative regulators of cell proliferation and metabolism in the fetus. Since the liver is a potential target tissue of these hormones during fetal life, we characterized the hepatic receptors for Sm-C/insulin like growth factor I (Sm-C/IGF-I) and insulin during the second trimester of human fetal development. Membrane-enriched fetal liver homogenates specifically bound 8.9 +/- 1.5% (+/- SD) of added [125I]insulin and 5.1-7.2% of [125I]Sm-C/IGF-I. Binding of both hormones was constant from 12-20 weeks gestation and was much greater than that in adult liver membranes. Analysis of dose-response data indicated high affinity between each receptor and its respective ligand (Kd for the Sm-C/IGF-I receptor, 2.2 X 10(-10) M; Kd for insulin receptor, 5.2 X 10(-10) and 7.7 X 10(-9) M). Limited cross-reactivity (approximately 1:1,000) of insulin with the Sm-C/IGF-I receptor and Sm-C/IGF-I with the insulin receptor was found. Affinity labeling studies showed that each receptor possessed an approximately 135,000-dalton subunit which was a part of a larger disulfide-linked complex. Thus, the human fetal liver has specific receptors for Sm-C/IGF-I and insulin that are similar to those described for other tissues in terms of both hormone-binding characteristics and subunit structure, suggesting that these receptors mediate important cellular functions at this stage of fetal development.

Growth hormone therapy of Turner syndrome: the impact of age of estrogen replacement on final height. Genentech, Inc., Collaborative Study Group.

Clinical trials of recombinant human GH therapy in Turner syndrome that began more than a decade ago show that GH accelerates the linear growth rate. Several studies indicate that final height is also improved, although the magnitude of the increase has been debated. The age at which feminization is induced could be an important factor in determining the patient's ultimate growth response. To test this, 60 patients from a large (n = 117), previously unreported, clinical trial of GH treatment were randomly assigned to begin conjugated estrogens at either 12 or 15 yr of age. The 60 patients were all less than 11 yr of age at entry (mean, 9.5 yr) and received 0.375 mg/kg x week of GH for nearly 6 yr on a daily or three times weekly regimen. Height gain was calculated by comparing the study patients' final or near final heights to their pretreatment projected heights as well as to those of a separate set of age-matched, historical control patients. Patients in whom estrogen treatment was delayed until age 15 yr gained an average of 8.4 +/- 4.3 cm over their projected height, whereas those starting estrogen at 12 yr gained only 5.1 +/- 3.6 cm, on the average (P < 0.01). Analysis of the interval data showed that growth was stimulated for approximately 2 yr after estrogen initiation, but then declined in association with bone age advancement. Patients who were older than 11 yr at entry (n = 57) all initiated estrogen 1 yr after beginning GH and showed a mean gain in adult height of 4.7 cm, similar to those given estrogen at age 12 yr. Multivariate analysis revealed that the number of years of GH therapy before estrogen treatment was a strong factor in predicting height gained, indicating that the timing of estrogen introduction is an important determinant of final height in this cohort of GH-treated patients with Turner syndrome matched for baseline age and other characteristics.

Growth hormone insensitivity associated with elevated circulating growth hormone-binding protein in children with Alagille syndrome and short stature.

The purpose of this study was to assess GH sensitivity in children with Alagille syndrome (syndromic intrahepatic bile duct paucity) by examining their response to short term administration of recombinant human GH (rhGH). Serum levels of insulin-like growth factor-I (IGF-I) were low despite elevated overnight integrated serum GH concentrations. Administration of rhGH (0.05 mg/kg.day for 3 days) to four growth-retarded children with Alagille syndrome did not significantly alter the serum concentrations of IGF-I and insulin, blood urea nitrogen, or urinary calcium excretion. In contrast, circulating IGF-I increased 2-fold in two children with Alagille syndrome and normal stature. In the control group, consisting of seven prepubertal children with GH deficiency, the predicted changes in response to rhGH in serum concentrations of IGF-I and insulin, urea nitrogen, and urinary calcium excretion were observed. Serum GH-binding protein levels, measured by a ligand-mediated immunofunctional assay, were significantly higher in children with Alagille syndrome than in children with cirrhosis or GH deficiency. We conclude that growth-retarded children with Alagille syndrome are insensitive to GH. The growth disturbances and metabolic defects may be due in part to failure to increase IGF-I concentrations in response to GH, implying that growth-retarded children with Alagille syndrome may benefit from IGF-I treatment.