Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I.

Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-II, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF-I on fibroblasts. When IGFBP-5 was present only in the medium, it was degraded to a 22-kD fragment and had no effect on IGF-I-stimulated growth. We conclude that IGFBP-5 is present in fibroblast ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular growth response to these growth factors.

An intronic growth hormone receptor mutation causing activation of a pseudoexon is associated with a broad spectrum of growth hormone insensitivity phenotypes.

CONTEXT: Inherited GH insensitivity (GHI) is usually caused by mutations in the GH receptor (GHR). Patients present with short stature associated with high GH and low IGF-I levels and may have midfacial hypoplasia (typical Laron syndrome facial features). We previously described four mildly affected GHI patients with an intronic mutation in the GHR gene (A(-1)-->G(-1) substitution in intron 6), resulting in the activation of a pseudoexon (6Psi) and inclusion of 36 amino acids. OBJECTIVE: The study aimed to analyze the clinical and genetic characteristics of additional GHI patients with the pseudoexon (6Psi) mutation. DESIGN/PATIENTS: Auxological, biochemical, genetic, and haplotype data from seven patients with severe short stature and biochemical evidence of GHI were assessed. MAIN OUTCOME MEASURES: We assessed genotype-phenotype relationship. RESULTS: One patient belongs to the same extended family, previously reported. She has normal facial features, and her IGF-I levels are in the low-normal range for age. The six unrelated patients, four of whom have typical Laron syndrome facial features, have heights ranging from -3.3 to -6.0 sd and IGF-I levels that vary from normal to undetectable. We hypothesize that the marked difference in biochemical and clinical phenotypes might be caused by variations in the splicing efficiency of the pseudoexon. CONCLUSIONS: Activation of the pseudoexon in the GHR gene can lead to a variety of GHI phenotypes. Therefore, screening for the presence of this mutation should be performed in all GHI patients without mutations in the coding exons.

Growth hormone receptor gene mutations in two Italian patients with Laron Syndrome.

Laron Syndrome (LS) represents a condition characterized by GH insensitivity caused by molecular defects in the GH receptor (GHR) gene or in the post-receptor signalling pathway. We report the molecular characterization of two unrelated Italian girls from Sicily diagnosed with LS. The DNA sequencing of the GHR gene revealed the presence of different nonsense mutations, occurring in the same background haplotype. The molecular defects occurred in the extracellular domain of the GHR leading to a premature termination signal and to a truncated non-functional receptor. In one patient, a homozygous G to T transversion, in exon 6, led to the mutation GAA to TAA at codon 180 (E180X), while in the second patient a homozygous C to T transition in exon 7 was detected, causing the CGA to TAA substitution at codon 217 (R217X). Both probands presented the polymorphisms Gly168Gly and Ile544Leu in a homozygous state in exons 6 and 10, respectively. The E180X represents a novel defect of the GHR gene, while the R217X mutation has been previously reported in several patients from different ethnic backgrounds but all from countries located in the Mediterranean and Middle Eastern region.

Excellent growth response to growth hormone therapy in a child with PTPN11-negative Noonan syndrome and features of growth hormone resistance.

We report a child with Noonan syndrome, referred with severe short stature (height--5.4 SD) and biochemical features of GH resistance. The Noonan syndrome phenotype was confirmed by a clinical geneticist, however analysis of the protein tyrosine phosphatase nonreceptor type 11 (PTPN11) gene showed no mutation. Baseline serum IGF-I, IGFbinding protein 3 (IGFBP-3) and acid-labile subunit (ALS) were low, and in an IGF-I generation test, IGF-I did not increase into the normal range and IGFBP-3 and ALS did not change. These results are consistent with GH resistance. Treatment with human GH (hGH) was given in a dose of 0.05 mg/kg/day and height velocity increased from 5.6 to 10.7 cm/yr during the first year, and 8.9 cm/yr during the second year of therapy. Height standard deviation score has increased by 1.85 after 2 and a half yr of therapy. Serum IGF-I, IGFBP-3 and ALS values increased well into the normal range. This case shows that the potential value of GH therapy must be evaluated in each patient individually and that an excellent response may occur in a child with a PTPN11-negative genotype.

IGFs and IGFBPs in GH insensitivity.

IGF-I, IGFBP-3 and ALS are GH-dependent peptides and their production is disturbed in states of GH insensitivity. This chapter explores the relative degrees of IGF-I, IGFBP-3 and ALS deficiency across the spectrum of GH insensitivity. In classical GH insensitivity syndrome (GHIS), known as Laron syndrome, due to GH receptor (GHR) deficiency, serum IGF-I, IGFBP-3 and ALS are severely reduced with inability to produce these peptides during an IGF-I generation test. Across the spectrum of severity of GHR defects, some patients have short stature and normal facial appearance, so-called partial or non-classical GH insensitivity. In these cases the IGF-I, IGFBP-3 deficiency is less severe. A positive relationship exists between height SDS and IGFBP-3 SDS (r2 = 0.45, p < 0.001) in patients from the European series with GHIS. In a new series of GHIS cases (n = 36) there was a significant difference in IGFBP-3 and ALS (p < 0.05) between classical (n = 25) and non-classical cases (n = 11). IGF-I, IGFBP-3 and ALS were significantly higher (p < 0.05) in pubertal compared with pre-pubertal subjects in the same series. In idiopathic short stature (ISS), heterozygous mutations of the GHR may have a dominant negative effect. ISS patients have lower IGF-I levels than the normal population. In 21 cases, mean IGF-I SDS was -1.39 (-2.4 to -1.16) and IGFBP-3; -0.45 (-1.13 to 0.38). However, IGF-I and IGFBP-3 responses in the IGF-I generation test were generally normal. In acquired GHI due to chronic illness such as Crohn's disease, juvenile arthritis and cystic fibrosis, IGF-I deficiency is present, although IGFBP-3 is usually normal. In summary, assessment of IGF-I, IGFBP-3 and ALS contributes to diagnosis in GH insensitivity states. In our experience, IGF-I is more sensitive to disturbance of GH action that IGFBP-3, however in severe GHIS cases, IGF-I is usually undetectable and measurement of IGFBP-3 is valuable as a guide to the severity of the biological defect.

Regulation of insulin-like growth factor (IGF) binding proteins in transgenic mice with altered expression of growth hormone and IGF-I.

The insulin-like growth factors (IGFs) are present in extracellular fluids bound to specific, high affinity IGF binding proteins (IGFBPs). IGFBPs are believed to mediate IGF transport to tissues and to modulate their actions on target cells. To determine whether IGF-I can modulate IGFBP concentrations in blood and to distinguish the effects of IGF-I from those of GH, we assessed serum IGFBP concentrations in four genotypically distinct groups of sibling transgenic (Tg) mice that differed in respect to their expression of IGF-I and GH. This unique physiological situation was created by crossing IGF-I Tg mice to GH-deficient, dwarf mice in whom somatotrophs were genetically ablated by the expression of a diphtheria toxin transgene in the somatotrophs. Because both Tg mouse lines are hemizygous for their respective transgene, progeny of the cross differ genotypically, according to whether or not they carry one or both transgenes, and phenotypically in regard to their relative expression of IGF-I and GH. GH-deficient mice showed a 15.7-fold decrease in serum IGF-I and a 5.5-fold decrease in serum IGFBP-3, but no change in a serum doublet band of 29,000 to 34,000 Mr, as assessed by ligand blotting. When IGF-I was expressed in the GH-deficient mice, serum levels of IGF-I and IGFBP-3 were 69% and 64% of those in normal sera, respectively. The 29,000 to 34,000 Mr doublet bands also increased. The ternary 150 kilodalton IGF-IGFBP complex, however, was not restored, presumably because IGF-I has no influence on the expression of the acid-labile subunit in this complex. In mice with IGF-I overexpression, serum IGFBP-3 was increased 2.1-fold and the sum of the 29,000 to 34,000 doublet bands was increased 2.9-fold. Immunoblotting showed that the changes in the 29,000 to 34,000 Mr forms observed by ligand blotting appeared to be predominantly due to changes in IGFBP-2. The results show that IGF-I can induce IGFBP-3 and IGFBP-2 independently of GH and that IGF-I is a major controller of these binding proteins.

Insulin-like growth factor binding protein secretion by breast carcinoma cell lines: correlation with estrogen receptor status.

Breast tumor cell lines have been shown to secrete several distinct polypeptide growth factors, although conflicting results exist for the insulin-like growth factors (IGFs). In contrast a limited number of breast tumor cell lines have definitely been shown to secrete the high affinity IGF binding proteins (IGFBPs) that modify IGF actions. To characterize the types of IGFBPs that are secreted by breast tumor cell lines, conditioned medium was collected from seven separate tumor cell lines, three of which were estrogen receptor (ER) negative, and four of which were ER positive. All three of the ER negative cell lines, MDA-231, MDA-330, and HS578T, secreted binding proteins of 49,000 and 43,000 Mr (IGFBP-3) as well as 29,000 (IGFBP-1) and 24,000 Mr. In contrast, all four ER positive cell lines secreted 34,000 (IGFBP-2) or 24,000 Mr forms, and none secreted the 49,000 and 43,000 or 29,000 Mr forms. BT-20, a cell line that is positive for ER messenger RNA (mRNA) but negative for ER protein, secreted predominantly a 34,000 Mr protein. The amount of total IGFBP activity released in 24 h ranged between 0.4 and 5.6 nM equivalents of IGFBP-1, and there was no significant difference between the ER positive and negative cell lines. The MCF-7 cells that produced predominantly 34,000 and 24,000 Mr forms showed a 1.8-fold increase in IGFBP secretion after estrogen stimulation. Immunoblotting and a specific RIA for IGFBP-1 showed that only the ER negative lines MDA-330, MDA-231, and HS578T secreted this form. Northern blotting analysis for the mRNA encoding this protein showed that both MDA-330 and MDA-231 contained a single 1.6 kilobase mRNA species that hybridized with an IGFBP-1 complementary DNA (cDNA) probe. Immunoblotting analysis of the other cell lines showed that only the 34,000 Mr form secreted by the ER positive cell lines reacted with IGFBP-2 antisera. Exposure of the conditioned media from the three ER negative cell lines to N-glycanase revealed that the 49,000 and 43,000 Mr forms of IGFBP were glycosylated and therefore probably represent IGFBP-3. We conclude that ER negative cell lines secrete three forms of IGFBPs, IGFBP-1, IGFBP-3, and a 24,000 Mr form. In contrast, the ER positive cell lines secrete predominantly IGFBP-2 and the 24,000 Mr form but do not secrete IGFBP-3 or 1.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factor (IGF) binding to cell monolayers is directly modulated by the addition of IGF-binding proteins.

Insulin-like growth factor-I (IGF-I) binds to specific receptors and IGF-binding proteins (IGFBPs) that are present on cell surfaces. The analysis of [125I]IGF-I binding to human fibroblasts is complicated by IGFBPs on the cell surface and their release into the medium during the binding assay. This release alters the distribution of [125I]IGF-I between type I IGF receptors and both soluble as well as cell surface-associated IGFBPs. In the present study we have determined the effects of three different forms of IGFBPs on [125I]IGF-I binding to cell surface binding sites of human fetal fibroblasts (GM10 cells) and porcine smooth muscle cells. Human 29,000 mol wt (Mr; IGFBP-1), bovine 34,000 Mr (IGFBP-2), and bovine 46,000 Mr (IGFBP-3) forms of IGFBP were compared. Each of the three IGFBPs inhibited [125I]IGF-I binding to the cell surface of both cell types. This effect was due to increased binding of [125I]IGF-I by the IGFBPs in the assay buffer. At equimolar concentrations, IGFBP-3 was more effective than either IGFBP-1 or IGFBP-2 in blocking cell surface binding. The addition of increasing concentrations of unlabeled IGF-I in the presence of each IGFBP showed that either IGFBP-1 or IGFBP-3, but not IGFBP-2, resulted in a paradoxical increase in [125I]IGF-I binding to the cell surface. The paradoxical increase occurred in the presence of excess insulin, indicating that unsaturated type I IGF receptors are not required to demonstrate this phenomenon. In a physiological salt solution, the order of affinity of the IGFBPs for IGF-I was IGFBP-3 greater than IGFBP-1 greater than IGFBP-2. These differences in affinity appear to account for the differences in IGF-I competition for binding that are seen when each of the three proteins is added. Thus, IGFBPs have the potential to alter the partitioning of IGF-I between cell surface-associated IGFBPs, membrane receptors, and the IGFBPs in extracellular fluids. The various forms of IGFBP affect IGF cell surface binding differently, and therefore, each may have distinct effects on IGF target cell actions.

Regulation of serum insulin-like growth factor-I (IGF-I) and IGF binding proteins during rat pregnancy.

Serum concentrations of insulin-like growth factor-I (IGF-I) in rats are reduced dramatically in the latter half of pregnancy, decreasing from 1758 +/- 356 ng/ml at 12 days of pregnancy (mean +/- SD) to 761 +/- 192 ng/ml at 15 days. After parturition, IGF-I increases to nonpregnant values in 4 days. Using ligand blotting, we have demonstrated that most of the serum IGF binding proteins (IGFBPs) are concurrently reduced during pregnancy. IGFBP-3, the predominant IGFBP in nonpregnant serum, is reduced to 1.3% of nonpregnant values by 21 days of pregnancy and begins to rise within 1 h postpartum (PP). The sera of 21-day pregnant (but not nonpregnant) rats degrade IGFBP-3 in vitro, and this degradation is prevented by the protease inhibitor antipain. Decreased serum IGF-I concentrations during pregnancy, therefore, may result from reduced IGFBP-3 concentrations causing increased IGF-I clearance. In addition, steady state IGF-I mRNA and peptide levels in liver are decreased in 21-day pregnant rats (37% and 42% of 4 day PP levels, respectively), suggesting that decreased synthesis of IGF-I may also lead to lower serum IGF-I concentrations. After bolus injection, [125I]IGF-I is cleared from the serum of pregnant rats nearly 5 times faster than that of 4 day PP rats (1.21 vs. 0.25 ml/min/kg, respectively). Urinary clearance is relatively insignificant (less than 4%), and [125I]IGF-I does not cross the placenta. The intermediate distribution phase of IGF-I is slower in pregnant rats than in PP rats (t1/2 alpha, 17.1 vs. 5.4 min), whereas the terminal elimination of IGF-I is twice as fast (t1/2 beta, 228.1 vs. 106.4 min). The prolonged IGF-I distribution phase in the pregnant rats may result from decreased concentrations of 34,000 and 30,000 mol wt IGFBPs, which may transport IGF-I to tissues. The faster serum elimination half-life may result from diminished IGFBP-3, leading to greater IGF-I availability to tissues in pregnancy.

Circulating aldosterone levels are unexpectedly low in children with acute meningococcal disease.

The incidence of meningococcal disease in childhood has risen over the past decade. Mortality remains high for those who develop septic shock and purpura fulminans. Poor perfusion, hypotension, and loss of intravascular circulating volume may be expected to influence both mineralocorticoid and glucocorticoid secretion. The aim of the study was to define adrenocortical hormone status at presentation. Sixty children admitted to the pediatric intensive care unit were studied. Children were divided into two groups: group A (n = 31), with meningococcal sepsis, mean age 4.4 yr (range 0.5-14.4), predicted risk of mortality mean 32.3% (range 0.5-99.3%); and group B (n = 29), with other diagnoses (post major surgery and with severe respiratory infections), mean age 4.1 yr (range 0.3-16.3), predicted risk of mortality mean 9.4% (range 0.2-83%). The groups were not significantly different for age. Plasma levels of aldosterone and cortisol were determined by RIA. The mean plasma aldosterone concentration on admission in group A was 427.5 +/- 88.1 pg/ml, with 96.7% of values within the normal range for age for healthy children and were significantly lower than group B mean, 1489.2 +/- 244.2 pg/ml (P < 0.0001), with 59.3% of values above the normal range. In group A there was no correlation with plasma concentrations of sodium, potassium, or volume of colloid infused in the previous 8 h. In group A mean serum cortisol mean values were 799.5 +/- 75.9 nmol/liter and in group B cortisol levels were 703.4 +/- 78.6 nmol/liter (P = n.s.). We conclude that children with meningococcal disease present with lower plasma aldosterone concentrations than other patients in the pediatric intensive care unit, for which there is no clear explanation. Further work is needed to elucidate the mechanisms underlying this finding and to examine its clinical implications.

Effects of insulin-like growth factor I (IGF-I) therapy on body composition and insulin resistance in IGF-I gene deletion.

We have recently reported a patient with a homozygous partial deletion of the insulin-like growth factor-I (IGF-I) gene, resulting in IGF-I deficiency, insulin resistance, and short stature. Recombinant human IGF-I (rhIGF-I) therapy has been shown to improve insulin sensitivity (Si) and growth in other causes of IGF-I deficiency. We now report results of 1 yr of rhIGF-I therapy on body composition, bone mineral density (BMD), insulin sensitivity, and linear growth in this patient. rhIGF-I therapy was initiated at age 16.07 yr (bone age, 14.2 yr), at a starting dose of 40 microg/kg daily, increasing after 3 months to 80 microg/kg daily. Body composition, BMD, markers of bone mineralization, and auxological parameters (height, weight) were measured at 0, 6, and 12 months after start of therapy. Si, acute insulin response to glucose, and glucose effectiveness were determined at baseline, 3 months, and 12 months into therapy. On IGF-I therapy, body mass index increased from 17 kg/m2 to 18.6 kg/m2. Body composition studies (dual-energy x-ray absorbtiometry) revealed an initial decrease in total body fat, from 19.9% at baseline to 15.1% at 6 months; but by 12 months of therapy, this had reversed, with an increase to 21.8%. Si, calculated using Bergman's minimal model, was substantially reduced at baseline at 1.45 x 10-4 min-1 (microU/mL) [normal value, 5.1 x 10-4 min 1 (lean adult male)]. rhIGF-I therapy resulted in a dose-related improvement of Si into the normal range (NR) (rhIGF-I dose: 40 microg/kg x day, Si = 2.06 x 10-4 min-l; rhIGF-I dose: 80 microg/kg x day, Si = 4.39 x 10-4 min-1). Baseline reduction in Si was accompanied by elevated acute insulin response to glucose, which also fell in a dose-dependent manner. Baseline BMD was severely reduced when compared with age-matched controls (-4.88 SD); however, calculation of bone mineral apparent density indicated that the true reduction in BMD was minimal. rhIGF-I therapy increased BMD by 17% and bone mineral apparent density by 7%, indicating that IGF-I has a greater effect on bone growth than bone mineralization. Bone turnover markers also increased on rhIGF-I; mean serum osteocalcin: 8.3 ng/mL pretreatment, 21.7 ng/mL after 6 months of rhIGF-I (NR for adult male, 3.4-9.1 ng/mL); mean bone specific alkaline phosphatase: 36.5 U/L pretreatment, 82.2 U/L after 6 months of therapy (NR for adult male, 15-41). Height velocity increased from 3.8 cm/yr pretreatment to 7.3 cm/yr on 80 microg/kg.day of rhIGF-I. In this patient with severe insulin resistance, therapy with rhIGF-I resulted in beneficial effects on Si, body composition, bone size, and linear growth. These results have implications for IGF-I therapy in a variety insulin resistant states.

Effects of recombinant human insulin-like growth factor I (IGF-I) therapy on the growth hormone-IGF system of a patient with a partial IGF-I gene deletion.

We have previously reported a 17.2-yr-old boy with severe growth retardation and undetectable serum levels of insulin-like growth factor I (IGF-I) due to a partial deletion of the IGF-I gene. The aim of this study was to investigate the effects of recombinant human IGF-I (rhIGF-I) therapy on the GH-IGF system of this patient to gain further insights into its growth-promoting and metabolic actions. To assess the changes in GH, IGFs, IGF-binding proteins (IGFBPs), acid-labile subunit (ALS), and insulin levels, blood samples were obtained before therapy and during the first year of treatment. Hormones were analyzed by specific RIAs. Overnight GH profiles were performed before and at 1, 6, and 12 months of therapy. Fasting ALS, IGF-II, IGFBP-3, IGFBP-2, IGFBP-1, and insulin levels before rhIGF-I treatment were 46.3 mg/L, 1044 microg/L, 5.8 mg/L, 73 ng/mL, 4.7 ng/mL, and 27.3 mU/L, respectively. IGF-II, ALS, and insulin levels were elevated, whereas IGFBP-1 and IGFBP-2 levels were decreased compared to reference values. Twenty-four hours after a single s.c. injection of rhIGF-I (40 microg/kg), the concentrations were 46 mg/L, 888 microg/L, 6.9 mg/L, 112 ng/mL, 5.0 ng/mL, and 21.0 mU/L, respectively. After a single s.c. injection of rhIGF-I of 40 or 80 microg/kg x day and modelling the data using a two-compartment model, the half-lives of elimination were 15.7 and 14.3 h, with a maximum increase in IGF-I levels to 341 and 794 microg/L around 7 h, respectively. An increase in IGFBP-3 levels was observed with both doses of rhIGF-I, with a peak values of 9 mg/L. GH profiles showed a decrease in peak amplitude from 342 to 84 mU/L at 1 month, to 67 mU/L at 6 months, and to 40 mU/L at 1 yr of therapy, with no significant changes in peak number. A significant increase in IGFBP-1 levels was observed during treatment with 80 microg/kg x day IGF-I, reflecting the inhibitory effect of rhIGF-I on insulin secretion. The clinical response to rhIGF-I therapy was an increased height velocity from 3.8 cm/yr before treatment to 6.6 cm/yr. Increased lean body mass correlated with changes in the doses of rhIGF-I and, in turn, with the biochemical changes in the GH-IGF axis. Similar to healthy individuals, this patient had normal IGFBP-3 and ALS levels, which are the major regulators of the pharmacokinetics of rhIGF-I. In summary, rhIGF-I treatment has improved linear growth and insulin sensitivity in this patient by restoring IGF-I levels and by normalizing circulating GH, IGFBP, and insulin levels.

Growth hormone (GH) insensitivity syndrome due to a GH receptor truncated after Box1, resulting in isolated failure of STAT 5 signal transduction.

Congenital GH insensitivity syndrome (GHIS) is usually the result of a mutation in the extracellular domain of the GH receptor (GHR). We report one of only a small number of mutations so far identified within the intracellular domain of the GHR. The probands are a 53-yr-old woman, height 114 cm (SD score, -8.7), peak GH 45 microg/liter during hypoglycemia, IGF-I 8.0 microg/liter [normal range (N) N 54-389], IGF binding protein-3 16 nmol/liter (N 61-254), GHBP 6.8% (N > 10); and her 57-yr-old brother, height 140 cm (SD score, -6), IGF-I 38.8 micro g/liter (N 54-290), IGF binding protein-3 30 nmol/liter (N 61-196). Both patients were homozygous for a 22-bp deletion in the DNA encoding the cytoplasmic domain of the GHR, resulting in a frameshift and premature stop codon. The resultant GHR is truncated at amino acid 449 (GHR1-449) after Box1, the Janus kinase 2 binding domain of the receptor. Functional studies in HEK293 and Chinese hamster ovary cells show GHR1-449 to have a cellular distribution similar to that of the wild-type GHR, judged by binding of iodinated GH, FACS analysis, and immunocytochemistry. Western blot analysis showed GH-induced phosphorylation of Janus kinase 2, signal transducer and activator of transcription (Stat)3, and Erk2 for both GHR1-449 and wild-type GHR. However, no Stat5 activity was detected in cells expressing GHR1-449, consistent with the fact that GHR1-449 contains no Stat5 binding site. In conclusion, we report two adult siblings with GHIS due to a mutation in the intracellular domain of GHR resulting in a selective loss of Stat5 signaling. Results are consistent with the hypothesis that the loss of signaling through the Stat5 pathway results in GHIS.

Comparison of continuation or cessation of growth hormone (GH) therapy on body composition and metabolic status in adolescents with severe GH deficiency at completion of linear growth.

Although GH replacement improves the features of GH deficiency (GHD) in adults, it has yet to be established whether cessation of GH at completion of childhood growth results in adverse consequences for the adolescent with GHD. Effects of continuation or cessation of GH on body composition, insulin sensitivity, and lipid levels were studied in 24 adolescents (13 males, 11 females, aged 17.0 +/- 0.3, yr, mean +/- se, puberty stage 4 or 5) in whom height velocity was less than 2 cm/yr. Provocative testing confirmed severe GHD [peak GH < 9 mU/liter (3 microg/liter)] in all cases and was followed by a lead-in period of 3 months during which the pediatric dose of GH continued unchanged. Baseline investigations were then performed using dual-energy x-ray absorptiometry (body composition), lipid measurements, and assessment of insulin sensitivity by both homeostasis model assessment and a short insulin tolerance test. Twelve patients remained on GH (0.35 U/kg.wk), and 12 patients ceased GH treatment. The groups were followed up in parallel with repeat observations made after 6 and 12 months. No endocrine differences were evident between the groups at baseline. GH cessation resulted in a reduction of serum IGF-I Z score [-1.62 +/- 0.29, baseline vs. -2.52 +/- 0.12, 6 months (P < 0.05) vs. -2.52 +/- 0.10, 12 months (P < 0.01)] but values remained unchanged in those continuing GH replacement. Lean body mass increased by 2.5 +/- 0.5 kg ( approximately 6%) over 12 months in those receiving GH but was unchanged after GH discontinuation. Cessation of GH resulted in increased insulin sensitivity [short insulin tolerance test, 153 +/- 22 micromol/liter.min, baseline vs. 187 +/- 20, 6 months (P < 0.05) vs. 204 +/- 14, 12 months (P = 0.05)], but no significant change was seen during 12 months of GH continuation. Lipid levels remained unaltered in both groups. Continuation of GH at completion of linear growth resulted in ongoing accrual of lean body mass (LBM), whereas skeletal muscle mass remained static after GH cessation in these adolescents with GHD. This divergence of gain in LBM is of potential importance because increases in LBM occur as a feature of healthy late adolescent development. GH is a major mediator of insulin sensitivity, independent of body composition in adolescents. Further studies are required to determine whether discontinuation of GH in the adolescent with severe GHD once linear growth is complete results in long-term irreversible adverse physical and metabolic consequences and to determine conclusively the benefits of continuing GH therapy.

The effect of cessation of growth hormone (GH) therapy on bone mineral accretion in GH-deficient adolescents at the completion of linear growth.

In many countries, treatment of childhood-onset GH deficiency (GHD) with GH ceases when linear growth is complete. Peak bone mass occurs several years after the completion of linear growth. Given that GH has important anabolic actions on bone, discontinuation of GH therapy at the completion of linear growth may have adverse consequences for the attainment of peak bone mass in adolescent GHD patients. In this United Kingdom multicenter study, 24 adolescents (13 males, mean age 17.0 +/- 1.4 yr, SD) with severe GHD were randomized to discontinue or continue GH (0.35 IU/kg x wk) at the completion of linear growth. Whole body bone mineral content (BMC) and lumbar spine bone mineral density were assessed by dual-energy x-ray absorptiometry at baseline and then at 6-month intervals for 1 yr. Markers of bone remodeling (serum bone-specific alkaline phosphatase and urinary deoxypyridinoline) were measured at the same time points. In patients who continued GH (GH+), median BMC increased by 3.8% (interquartile range, 2.6, 5.9, P < 0.001) at 6 months; and by 6.0% (3.7-9.1, P < 0.001) at 12 months. In patients who discontinued GH (GH-) median BMC was unchanged at 6 and 12 months (+1.9%, -0.4-4.2, P = 0.9; and +2.4%, 0.4-4.9, P = 0.5, respectively, median, interquartile range). The differences in median change in BMC between the two groups at 6 and 12 months was marginally significant (P = 0.085 and 0.074, respectively). Mean lumbar spine bone mineral density increased by 4.7 (95% confidence interval, 1.0, 8.2) at 12 months in patients continuing GH (P = 0.01), but the mean change was not statistically significant change in patients who discontinued GH [+2.7% (95% confidence interval, -0.8, +6.2)]. These preliminary data suggest that, in adolescent patients with severe GHD, discontinuation of GH at completion of growth may limit the attainment of peak bone mass in this patient group. This may predispose to clinically significant osteopenia in later adult life.

The short-term effects of growth hormone therapy on height velocity and cardiac ventricular wall thickness in children with Noonan's syndrome.

Noonan's syndrome (NS) is associated with short stature and cardiac defects. Small studies reported linear growth increases with recombinant human GH (rhGH) therapy, but also raised concerns related to the anabolic effects of rhGH and the possible progression of ventricular hypertrophy. We report a multicenter study examining the efficacy and safety of rhGH (4 IU/m2.day, sc) in children with NS. Entry criteria were: NS confirmed by single observer, height SD score less than -2(UK Height Standards 1990), prepubertal, and normal maximal left ventricular (LV) wall thickness less than 1 cm by 2-dimensional echocardiography. Thirty subjects were recruited (19 males and 11 females), aged 8.9 +/- 0.5 yr (range, 4.8-13.7 yr). Growth was monitored for 12 months before and at 3-month intervals during therapy. Measurements of maximal LV wall thickness were taken at 0 and 12 months. Serum insulin-like growth factor I(IGF-I), IGF-II, and IGF-binding protein-3 levels were determined at 0, 3, 6, 9, and 12 months. Ten subjects with NS (4 females and 6 males), aged 8.8 +/- 0.7 yr (range, 6.3-11.8 yr), were monitored over the same period as a comparison group. In the treatment group, 27 subjects completed 12 months of therapy. Height SD score increased from -3.01 +/- 0.10 to -2.36 +/- 0.10 (P < 0.0001) after 12 months; height velocity (HV) increased from 4.9 +/- 0.2 to 8.9 +/- 0.3 cm/yr at 6 months and 8.1 +/- 0.4 cm/yr (P < 0.0001) from 6-12 months. The HV SD score increased from -0.7 +/- 0.15 to +2.42 +/- 0.32 over 12 months (P < 0.0001). The increase in HV was more than 2 cm/yr in 24 patients. IGF-I increased from 121 +/- 13 to 240 +/- 22 micrograms/L at 12 months (P < 0.0001), and IGF-binding protein-3 increased from 2.65 +/- 0.20 to 4.01 +/- 0.42 mg/L at 12 months (P = 0.0009). In the comparison group, there was no change in height SD score (-2.03 +/- 0.19), HV (4.4 +/- 0.24 CM/yr), or HV SD score (- 1.08 +/- 0.21). There was no increase in mean maximal LV wall thickness during the study in either the treatment group (12 month values were 0.63 +/- 0.02 cm at the mitral valve level and 0.66 +/- 0.02 cm at the papillary muscle level) or in the comparison group (0.63 +/- 0.04 cm at the mitral valve level and 0.61 +/- 0.03 cm at the papillary muscle level). In conclusion, rhGH was effective in 24 of the treated patients; these subjects achieved a significant increase in height SD score and HV over 1 yr. Abnormal anabolic effects of rhGH on myocardial thickness were not confirmed, and no patient developed features of hypertrophic cardiomyopathy.

Insulin-like growth factor I and the development of colorectal neoplasia in acromegaly.

Patients with acromegaly are at increased risk of colorectal neoplasia and, by analogy with high-risk nonacromegalic patients, may require regular colonoscopic screening. However, it is unknown whether the risk is equal in all patients or whether some should be regarded as carrying a particularly high risk. The aims of this study were: 1) to establish the natural history of colorectal neoplasia in acromegaly; 2) to establish which patients are at increased risk of developing neoplasia; and 3) to elucidate the influence of insulin-like growth factor I (IGF-I) in adenoma formation. A prospective colonoscopic evaluation of the development of new premalignant adenomas in the colon was performed in 66 patients with biochemically proven acromegaly who had previously undergone colonoscopic screening and removal of all visible polyps. Twenty-five patients (38%) had a total of 37 polyps detected at the second colonoscopy: nine (14%) had at least one adenoma, and 18 (27%) had one or more hyperplastic polyps (2 patients had both). The development of new adenomas, but not hyperplastic polyps, was associated both with elevated serum IGF-I (P < 0.005) and, to a lesser extent, with a previous adenoma at the original colonoscopy (P < 0.07). In summary, patients with acromegaly and in whom serum IGF-I remains elevated and/or who have had a previous adenoma should be regarded as having an especially high risk for the development of subsequent colorectal neoplasia. Serum IGF-I seems to be implicated in the development of colorectal neoplasia in acromegaly, although the exact mechanisms remain uncertain.

Optimizing growth hormone replacement therapy by dose titration in hypopituitary adults.

Although growth hormone (GH) replacement therapy is increasingly utilized in the management of adult hypopituitary patients, optimum dosing schedules are poorly defined. The use of weight-based or surface area-based dosing may result in overtreatment, and individual variation in susceptibility on the basis of gender and other factors is now being recognized. To optimize GH replacement and to explore further gender differences in susceptibility, we used a dose titration regimen, starting at the initiation of GH replacement therapy, in 50 consecutive adult-onset hypopituitary patients, and compared the results with those in 21 patients previously treated using a weight-based regimen. Titrated patients commenced GH 0.8 IU/day subcutaneously (0.4 IU/day if hypertensive or glucose tolerance impaired). Serum insulin-like growth factor I (IGF-I) was measured at 0, 2, 4, 6, 8, 10, and 12 weeks in all patients. Serum IGF binding protein 3 and acid labile subunit were measured at the same time points in 17 patients (8 male, 9 female). Patients were reviewed every 4 weeks and the dose of GH increased, if necessary, to achieve a serum IGF-I level between the median and the upper end of the age-related reference range. There was no significant difference between mean serum IGF-I at 2 and 4 weeks, or between 6 and 8 weeks, indicating that the full effects of a change in dose are evident within 2 weeks of that change. Maintenance doses were significantly higher in females than males [1.2 (0.8-2.0) vs. 0.8 (0.4-1.6) IU/day; median (range); P < 0.0001], and the median time to achieve maintenance dose was significantly shorter in males [4 (2-12) vs. 9 (2-26) weeks; P < 0.0001]. Median maintenance dose was lower overall than in a group of 21 patients initially commenced on GH using a weight-based dosing schedule, with subsequent adjustment of dose during clinical follow-up [1.5 (0.4-3.2) IU/day; P = 0.02]. Reduction in waist measurement and waist to hip ratio at 6 and 12 months was similar in females (P < 0.001) and males (P < 0.01). Well-being improved significantly after 3 months of GH therapy (14.2 +/- 5.9 vs. 7.4 +/- 4.5 SD; P < 0.0001), and there were no gender differences. Adult Growth Hormone Deficiency Assessment (AGHDA) scores at 6 months were similar to maintenance scores in patients commenced on weight-based regimens. Measurements of ALS and IGFBP-3 added no useful extra information to IGF-I in managing the dose titration. The practical scheme outlined for dose titration of GH replacement resulted in rapid achievement of lower maintenance doses than those achieved using conventional weight-based regimens without loss of efficacy. It was particularly important in female patients who demonstrated decreased overall sensitivity to GH and required higher doses to achieve the same effects as males. This constitutes the first report of a uniform titration regimen based on a defined target range of serum IGF-I in a large patient cohort.

Predictors of the outcome of surgical treatment in acromegaly and the value of the mean growth hormone day curve in assessing postoperative disease activity.

Acromegaly is associated with increased morbidity and mortality unless serum GH levels are persistently less than 5 mU/L ( approximately 2 ng/mL) after treatment. Transsphenoidal surgical resection is the best available treatment for restoring GH to such "safe" levels; however, criteria for the assessment of the response to treatment are not uniform. To determine the clinically most useful method of assessing disease activity postoperatively and identify predictors of a favorable response to surgical treatment, we have analyzed 67 patients with acromegaly who underwent transsphenoidal surgery between 1993 and 1998. We used three different definitions of a satisfactory or safe response: 1) a postoperative mean GH less than 5 mU/L obtained from averaging five serum GH values obtained throughout one day; 2) a random single GH less than 5 mU/L; or 3) a serum insulin-like growth factor I (IGF-I) level within the normal range. Relying on a single GH measurement alone, 9 of the 23 patients with a single postoperative mean GH level less than 5 mU/L obtained at least one GH value of more than 5 mU/L (false positive rate, 28%) and 8 of the patients with a postoperative mean GH value of more than 5 mU/L obtained a single GH value of less than 5 mU/L (false negative rate, 15%). Postoperatively, a significant increase in the fluctuation of random GH values around the mean was observed in patients who were rendered safe (coefficient of variation, from 26 +/- 2% to 53 +/- 6%; P < 0.001) compared with patients with persistence of inadequately controlled disease. However, 13% (3 of 23) of patients with mean postoperative GH levels of less than 5 mU/L had elevated serum IGF-I levels postoperatively, and 17% (8 of 44) of patients with mean serum GH levels more than 5 mU/L had postoperative IGF-I levels within the normal range. There was no difference in the rate of agreement between mean GH less than 5 mU/L and normalization of IGF-I in relation to the interval since operation when IGF-I levels were measured. Preoperative tumor size and pretreatment mean GH levels were the major determinants of the outcome of surgery, as patients who were rendered safe had significantly lower preoperative mean GH levels than patients who were not cured (median, 31 mU/L vs. 78.5 mU/L, P < 0.01). IGF-I levels were weakly correlated with tumor size and could not be used to predict the patients who would be rendered safe. Preoperative PRL levels were higher in patients who failed to achieve a surgical satisfactory outcome [498 mU/L (187-857) vs. 196 mU/L (136-315), P < 0.01]. In summary, although single random GH values and IGF-I values are both significantly correlated with mean GH levels, they should not be used as an alternative to averaging several GH values to assess disease activity, because of the pulsatile nature of GH secretion and the multiple factors that may influence serum IGF-I. Because significant discrepancies occur, particularly postoperatively, mean GH levels remain the more reliable indicator of surgical outcome and disease activity. As there is considerably more evidence relating long-term prognosis to serum GH levels than to IGF-I and discrepancies occur between GH levels and IGF-I, we suggest that mean serum GH levels and single IGF-I levels, measured early in the postoperative period, are currently the best biochemical guide to the adequacy of surgery and, hence, the need for further treatment.