Genetic analysis of short children with apparent growth hormone insensitivity.

BACKGROUND/AIMS: In short children, a low IGF-I and normal GH secretion may be associated with various monogenic causes, but their prevalence is unknown. We aimed at testing GH1, GHR, STAT5B, IGF1, and IGFALS in children with GH insensitivity. SUBJECTS AND METHODS: Patients were divided into three groups: group 1 (height SDS <-2.5, IGF-I <-2 SDS, n = 9), group 2 (height SDS -2.5 to -1.9, IGF-I <-2 SDS, n = 6) and group 3 (height SDS <-1.9, IGF-I -2 to 0 SDS, n = 21). An IGF-I generation test was performed in 11 patients. Genomic DNA was used for direct sequencing, multiplex ligation-dependent probe amplification and whole-genome SNP array analysis. RESULTS: Three patients in group 1 had two novel heterozygous STAT5B mutations, in two combined with novel IGFALS variants. In groups 2 and 3 the association between genetic variants and short stature was uncertain. The IGF-I generation test was not predictive for the growth response to GH treatment. CONCLUSION: In severely short children with IGF-I deficiency, genetic assessment is advised. Heterozygous STAT5B mutations, with or without heterozygous IGFALS defects, may be associated with GH insensitivity. In children with less severe short stature or IGF-I deficiency, functional variants are rare.

Acid-labile subunit deficiency: phenotypic similarities and differences between human and mouse.

IGF-I and IGF-II (IGFs) form higher molecular weight complexes with specific binding proteins (IGFBP-1 to -6). These complexes are referred to as binary complexes consisting of IGF-I or IGF-II and one IGFBP, or as ternary complexes each consisting of either of IGF-I or IGF-II, IGFBP-3 or -5, and an acid-labile subunit known as ALS. Ternary complex formation restricts the IGFs to the circulation and prolongs their half-life. Recently, the development of an animal model for ALS deficiency (the ALS-KO mouse) and the identification of a patient with an inactivating mutation in the IGFALS gene have provided the opportunity to assess the physiological role of this protein in the circulating IGF system. ALS deficiency has no effect on fetal growth in both the ALS-KO mice and the ALS-deficient patients. A modest reduction in post-natal growth in the null ALS mice and in the ALS-deficient patients was observed. The plasma concentrations of IGF-I and IGFBP-3 were markedly reduced both in ALS-KO mice and in the ALS-deficient patients. Basal GH levels remained normal in the ALS-KO mice and moderately increased in the ALS-deficient patients. Insulin-resistance was present in the ALS-deficient patients but not in the ALS-KO mice. Reduced bone mineral density (BMD) was present in mice and human ALS deficiency. Phenotypic features of complete ALS deficiency, that are very similar in mouse and human, include: a) the inability to form ternary complex, b) the small growth impairment in spite of the marked reduction in circulating IGF-I, and c) the reduction in BMD. On the other hand, insulin resistance and pubertal delay were observed only in human ALS deficiency. These findings underlie the important physiological role of ALS in the maintenance of the circulating IGF-I reservoir. Both models will be useful in identifying the respective roles of plasma and locally derived IGF-I in regulating metabolism and growth of specific tissues.

Estrogen priming effect on growth hormone (GH) provocative test: a useful tool for the diagnosis of GH deficiency.

We have studied the effect of estradiol (E2) on the GH-insulin-like growth factor (GH-IGF) axis in 15 prepubertal GH deficiency (GHD) children and 44 prepubertal or early pubertal children with idiopathic short stature (SS). All of them received a daily dose of micronized E2 (1 or 2 mg) or placebo, for 3 days, before a sequential arginine-clonidine test. In SS children, GH maximal responses were 17.8+/-10.9 on placebo and 27.9+/-14.5 microg/L on estrogen (P < 0.0001). The lower 95% confidence limits for GH maximal response changed from 3.7 microg/L (without E2) to 8.3 microg/L (on E2). In GHD children, no significant stimulatory effect of estrogen on GH levels was observed. After placebo, a cut-off limit of 3.7 microg/L (the lower 95% confidence interval limit) resulted in 73% sensitivity, 95% specificity, and an overall 90% diagnostic efficiency. After E2, a cut-off limit of 8.3 microg/L resulted in a sensitivity of 87%, a specificity of 98%, and a diagnostic efficiency of 95%. After placebo, 68% of SS showed normal IGF-I levels, and the mean did not change on E2 (13.7+/-6.3 vs. 14.3+/-6.8 nmol/L, not significant). In 93% of SS, IGF binding protein (IGFBP)-3 levels were normal during placebo. On E2, mean IGFBP-3 did not change (2.63+/-0.70 vs. 2.70+/-0.70 mg/L, not significant). In 14 of 15 GHD patients, IGF-I values were below normal on placebo, and the mean of the group did not change after E2. During placebo, 13 of 15 GHD children presented low IGFBP-3 values. During E2, there was a small significant increase in IGFBP-3 values (1.06+/-0.58 vs. 1.20+/-0.69 mg/L, P < 0.02). The highest diagnostic efficiencies for IGF-I and IGFBP-3 were observed during placebo (75% and 91%, respectively). We conclude that GH stimulation tests after E2 priming had the highest diagnostic efficiency. Our findings suggest that the effect of estrogen priming on GH stimulated levels, by reducing the number of false nonresponders, might be useful to better discriminate between normal and abnormal GH status in SS children.

Effect of deflazacort versus methylprednisone on growth, body composition, lipid profile, and bone mass after renal transplantation. The Deflazacort Study Group.

Kidney function, growth velocity, weight/height ratio, body composition, lipid profile, and bone mass were studied in a randomized, multicenter trial of deflazacort versus methylprednisone in 27 prepubertal patients with kidney transplantation. Methylprednisone (0.20+/-0.03) was replaced by deflazacort (13 patients, 0.30+/-0.03 mg/kg per day). After 12 months, creatinine clearance decreased significantly only during methylprednisone therapy. Growth velocity increased only in patients treated with deflazacort from 3.3+/-0.6 to 5.6+/-0.5 cm/year. Serum levels of several components of the insulin-like growth factor axis did not change. Weight/height ratio was increased in methylprednisone-treated patients (P<0.05) and decreased in deflazacort-treated patients (P<0.005). Lean body mass increased in both groups (P<0.005). Fat body mass and serum leptin increased only in methylprednisone-treated patients (P<0.025). Total cholesterol and low-density lipoprotein-cholesterol increased in methylprednisone-treated patients by 9.9% (P<0.05) and 12.5% (P<0.025). High-density lipoprotein-cholesterol increased by 21% (P<0.005) and apolipoprotein B decreased by 11% (P<0.005) in deflazacort-treated patients. Total skeleton and lumbar spine bone mineral density decreased in both groups, but at 1 year methylprednisone-treated patients had lost 50% more bone. Bone mineral content decreased only in methylprednisone-treated patients (P<0.01). Our data suggest that substituting deflazacort for maintenance methylprednisone might prevent height loss, excessive bone loss, and fat accumulation; and leads to an improvement in the lipoproteins of these children.

[Early diagnosis of multiple endocrine neoplasia type 2 (MEN 2) by detection of mutated RET proto-oncogene carriers]. / Diagnóstico precoz de neoplasia endocrina múltiple tipo 2 (MEN 2) por la deteccion de portadores de mutaciones en el proto-oncogen RET]

RET proto-oncogene mutation results in a dominant autosomic inherited syndrome (MEN 2) presenting three distinct subtypes: MEN 2A, MEN 2B, and familial medullary thyroid carcinoma (FMTC). Detection of RET proto-oncogene mutation is a predictor before clinical or biochemical evidence of the disease is present and leads to preventive thyroid removal since there is no effective treatment for metastases. The aim of the present study was to characterize mutations in the RET proto-oncogene in affected patients and to identify potential carriers in their families. Two families with FMTC (5 and 6 members), 4 with MEN 2A (5, 5, 4 and 3 members) and 2 with MEN 2B (5 and 1 members), were studied. DNA was obtained from blood samples in all patients and from thyroid or from pheonochromocytoma tissues in patients submitted to surgery. PCR amplification was performed using specific primers for exons 10, 11 and 16, followed by direct sequencing. Mutations at codon 634 in exon 11 were found in 16 subjects with FMTC and MEN 2A: TGC --> CGC (cysteine to arginine) in 9 cases, TGC --> TAC (cysteine to tyrosine) in 3, and TGC --> TTC (cysteine to phenilalanine) in 4. A unique mutation of codon 918 in exon 16, ATG --> ACG (methionine to threonine), was found in both MEN 2B affected patients. The mutations detected in DNA from peripheral blood were the same as those present in DNA extracted from tumor material. RET mutations were detected in all affected patients, confirming the diagnosis, and in 10 members of their families. In five of the carriers total thyroidectomy was performed. Anatomopathological study showed C-cells hyperplasia or in-situ microcarcinoma in two children (9 and 12 y) with no clinical signs of diseases and medullary thyroid carcinoma in three adults, who were previously unaware of the presence of thyroid nodules. The early detection of RET mutation followed by total thyroidectomy may prevent the development of the disease, specially in affected families, and avoid the fatal outcome of delayed medullary thyroid carcinoma diagnosis.

Differential expression of renal growth hormone receptor and its binding protein in experimental diabetes mellitus.

Growth hormone (GH) may have a role in the development of diabetic nephropathy. The effect of experimental diabetes on renal expression of the growth hormone receptor gene products, including the receptor itself (GHR) and its binding protein (GHBP) was examined. Adult female rats received i.v. streptozotocin and were killed at 7, 30, 90 and 180 days after the induction of diabetes. Diabetic animals had a pronounced increase in kidney weight and progressive albuminuria. In renal cortex, no change was seen in GHR mRNA levels throughout the observation period of 6 months, while a significant increase in cortical GHBP mRNA levels was observed after 1 month of diabetes and sustained for the rest of the study period. Immunohistochemical analysis of kidney sections revealed a stronger staining for GHBP at the cortical and inner medullary areas in the diabetic animals. These data indicate that although the GHR and GHBP mRNAs originate from the same gene, their renal levels are differentially regulated during the development of experimental diabetic kidney disease, suggesting a functional role for GHBP.

Persistence of autonomous ovarian activity after discontinuation of therapy for precocious puberty in McCune-Albright syndrome.

OBJECTIVE: The aim of this study was to evaluate the possibility of persistence of autonomous ovarian activity in girls with McCune-Albright syndrome (MAS) after withdrawal of medroxyprogesterone therapy administered for precocious puberty. DESIGN, SETTING, AND PARTICIPANTS: Five girls with MAS were followed-up 1.2 to 8.5 years after the end of treatment. The girls underwent luteinizing hormone-releasing hormone (LH-RH) tests, estradiol (E2) basal measurement, and pelvic ultrasound two times in the follow-up period. RESULTS: Menses resumed in four of five girls, 1.4 +/- 0.9 years after the end of treatment, at chronologic age of 11.3 +/- 1.3 years. Cycles for all girls were irregular. Three patients presented inadequate E2 levels (from 56 to 320 pg/mL) associated with low or absent gonadotropin response to LH-RH tests. The pelvic ultrasound showed ovarian cysts at the time of the study. CONCLUSION: These hormonal and ultrasonographic findings provide evidence of persistence of autonomous ovarian activity in some young women with MAS.

Decreased hepatic insulin-like growth factor (IGF)-I and increased IGF binding protein-1 and -2 gene expression in experimental uremia.

The imbalance between normal insulin-like growth factor-I (IGF-I) and markedly increased IGF binding protein (IGFBP) plasma levels plays a pathogenic role for growth retardation and catabolism in children with chronic renal failure. To investigate the mechanism of these alterations, experiments were performed in an experimental model of uremia in rats (5/6 nephrectomy) and in pair-fed and ad libitum-fed sham-operated controls Using a specific solution hybridization/RNase protection assay, we observed a marked reduction of hepatic IGF-I messenger RNA (mRNA) abundance at steady state in uremic animals (37 +/- 5% of control) compared both with pair-fed (65 +/- 10%) and ad libitum-fed controls (100 +/- 11%) (P < 0.001). Reduced IGF-I gene expression was clearly organ-specific; it was most pronounced in liver (significant vs., pair-fed controls) and lung and muscle tissue (significant vs., ad libitum-fed controls); no change was observed in kidney and heart tissue. To determine a potential mechanism of reduced hepatic IGF-I gene expression in uremia, the hepatic GH receptor gene expression in the same experimental animals was analyzed by specific solution hybridization/RNase protection assay. Uremic animals had a 20-30% reduction of hepatic GH receptor mRNA abundance compared with controls. Hepatic GHBP expression in uremia was decreased in parallel. Despite the reduction of hepatic IGF-I mRNA abundance, plasma IGF-I levels in uremia were not different from ad libitum-fed controls. This discrepancy is explained by an increased concentration of IGFBPs in uremic plasma. By RIA, plasma IGFBP-1 levels in uremia were increased 4-fold; by Western immunoblot, plasma IGFBP-2 levels were increased 7-fold and plasma IGFBP-4 levels were increased 2-fold compared with both control groups. Intact IGFBP-3 (M(r), approximately 48 kDa) and low molecular IGFBP-3 fragments were not significantly different among the three groups. By Northern blot analysis, hepatic IGFBP-1 mRNA levels in uremia were 2-fold higher than in controls. IGFBP-2 mRNA abundance in liver tissue was increased 4-fold, whereas in kidney there was a significant reduction of IGFBP-2 mRNA (30% of control). IGFBP-4 mRNA was increased by 50% in kidney but not in liver. Plasma insulin and corticosterone levels were not different among the groups. Our study shows that hepatic IGF-I gene expression was specifically reduced in uremia, partially as the consequence of a reduced hepatic GH receptor gene expression. One of the mechanisms contributing to increased IGFBP levels in uremia is increased hepatic gene expression of IGFBP-1 and IGFBP-2. The imbalance between reduced hepatic IGF-I production and increased hepatic IGFBP-1 and 2 production is likely to play a pathogenic role for catabolism and growth failure in CRF.

Developmental changes and differential regulation by testosterone and estradiol of growth hormone receptor expression in the rabbit.

To investigate the effects of testosterone and estradiol (E2) on growth hormone receptor (GH-R) gene expression, we measured GH-R mRNA levels in relation to the changes of sex steroid concentrations in the normal male rabbits aged 1-12 months and after administration of testosterone or E2 to castrated male rabbits. In the normal animals, E2 levels were below the detection limit in all age groups, and testosterone levels were below the detection limit at 1 month, increased at 2 months and reached the plateau of the adult levels after 4 months. Liver GH-R mRNA levels were low at 1 month, reached a peak at 2 months and then decreased slightly thereafter. In the castrated animals, liver and growth plate GH-R mRNA levels were increased in the testosterone-treated group (162.0 +/- 12.0%, p < 0.025; 128.4 +/- 7.6%; p < 0.025) and reduced in the E2-treated group (29.6 +/- 6.2%, p < 0.005; 53.6 +/- 11.3%, p < 0.025). Sex steroid administration did not result in any significant change in GH-R mRNA levels in striated muscle, kidney and heart. Serum GH concentrations were increased in E2 (15.3 +/- 7.7 microg/l vs 4.8 +/- 2.2 microg/l, p < 0.025) but the increase was not significant in testosterone-treated animals (8.4 +/- 7.7 microg/l vs 4.8 +/- 2.2 microg/l). Both testosterone and E2 treatment resulted in a reduction of mean serum growth hormone-binding protein (GHBP) levels compared to control animals (1077 +/- 422 pmol/l, p < 0.01; 1137 +/- 443 pmol/l, p < 0.01; 2308 +/- 565 pmol/l). We conclude that in addition to their stimulatory effect on GH secretion, testosterone and E2 have opposite effects on GH-R gene expression in liver and growth plate in the rabbit. The modulation of GH-R expression by sex steroids may be important for growth during sexual maturation in mammals.

The role of growth hormone, insulin-like growth factors (IGFs), and IGF-binding proteins in experimental diabetic kidney disease.

Early renal changes in type I diabetes are characterized by an increase in renal size, glomerular volume, and kidney function, and later by development of mesangial proliferation, accumulation of glomerular extracellular matrix, and increased urinary albumin excretion (UAE). Growth hormone (GH) and insulin-like growth factors (IGFs) have a long and distinguished history in diabetes mellitus, with possible participation in the development of long-term complications. In experimental diabetes in dwarf rats with isolated GH and IGF-I deficiency, a slower and lesser renal and glomerular hypertrophy is observed as compared with diabetic control animals with intact pituitary. Furthermore, diabetic dwarf rats with a diabetes duration of 6 months display a smaller increase in UAE, indicating that GH and IGF-I may be involved in the development of diabetic kidney changes. In line with this, administration of octreotide to streptozotocin (STZ)-diabetic animals with normal pituitary inhibits initial renal growth without affecting blood glucose levels, and 6 months' administration of octreotide to diabetic rats reduces long-term renal/glomerular hypertrophy and UAE. In addition, the initial increase in renal size and function in experimental diabetes is preceded by an increase in renal IGF-I, IGF-binding proteins (IGFBPs), and IGF-II/mannose-6-phosphate receptor (IGF-II/Man-6-P receptor) concentration. Finally, specific changes occur in renal GH-binding protein (GHBP) mRNA, IGF-I receptor mRNA, and IGFBP mRNA expression in long-term diabetes. In conclusion, the knowledge we have today indicates that GH and IGFs, through a complex system consisting of GHBP, IGFs, IGF receptors, and IGFBPs, may be responsible for both early and late renal changes in experimental diabetes.

Expression of insulin-like growth factor binding proteins in the rat kidney: effects of long-term diabetes.

Recent studies have shown that the renal synthesis of insulin-like growth factor binding proteins (IGFBPs) is altered in insulin-deficient diabetes mellitus, suggesting that these changes may be implicated in the alterations in renal function and morphology that accompany diabetes. To investigate the time course and the precise cellular distribution of changes in IGFBP expression, we used quantitative in situ hybridization to analyze renal IGF-I and IGFBP-1 to -5 messenger RNA (mRNA) localization and levels from 2 days to 6 months after the onset of streptozotocin-induced diabetes. There was an immediate sharp decline in IGF-I mRNA levels in the outer medulla that persisted for up to 3 months and a much smaller reduction in IGF-I mRNA levels in the medullary thick ascending limbs (MTALs). In nondiabetic animals, IGFBP-1 mRNA is most abundant in the MTALs. Immediately after the induction of diabetes, however, there was a greater than 2-fold increase in cortical IGFBP-1 mRNA and a 75% decrease in IGFBP-1 mRNA in MTALs. These changes persisted for up to 6 months in the diabetic animals. In contrast, IGFBP-5 mRNA levels were increased in the outer medulla and decreased in the cortex of diabetic kidneys. No significant changes in renal IG-FBP-2 mRNA levels or distribution were noted, and changes in IG-FBP-3 and -4 mRNA levels were subtle. In summary, streptozotocin-induced diabetes is associated with very prominent and complex alterations in renal IGF system gene expression, including robust increases in cortical IGFBP-1 and profound decreases in cortical IG-FBP-5 mRNA and medullary IGF-I mRNA levels. The divergent changes in IGFBP-1 and -5 mRNA levels in cortex vs. outer medulla indicate that regulation of IGFBP mRNA levels is quite complex.

Rat growth hormone receptor/growth hormone-binding protein mRNAs with divergent 5'-untranslated regions are expressed in a tissue-specific manner.

In the rat, the growth hormone receptor (GH-R) gene generates two transcripts, one encoding the transmembrane GH-R, and a shorter one encoding the GH-binding protein (GH-BP). These transcripts exhibit a high degree of heterogeneity in their 5'-untranslated regions (5'-UTRs). Some of the exons encoding these 5'-UTR variants may be flanked by distinct promoter regions whose activity would result in the tissue-specific expression of the GH-R gene. To assess this possibility, we used single-sided polymerase chain reaction (PCR) amplification to characterize 5'-UTR variants in rat GH-R cDNAs, and by using 5'-UTR-specific probes, we determined their pattern of expression in several tissues. Besides two previously described variants (V1 and V2), three new 5'-UTR variants were identified, extending 56 nucleotides (V3), 135 nucleotides (V4), and 209 nucleotides (V5) upstream of the ATG translation initiation codon. The expression of GH-R and GH-BP transcripts was clearly tissue specific. In the liver, GH-BP mRNA was the predominant transcript, whereas in other tissues, there was equivalent expression of both transcripts or predominant expression of GH-R mRNA. With respect to the tissue distribution of the 5'-UTR variants in particular, variants V1 and V5 exhibited a pattern of expression closely resembling that seen with an exon 2 probe, with the overall expression of variant V1 being much higher than that of variant V5. The V2 species was exclusively expressed in liver. Variant V3 was expressed at low levels in liver, muscle, heart, and kidney; in muscle and heart, it was preferentially associated with GH-BP transcripts. Variant V4, although present in liver, was more abundant in extrahepatic tissues and predominantly found in GH-R mRNA transcripts. Southern blot analyses were consistent with exon 2 and the exons encoding the V1 and V2 sequences being in proximity, with the other 5'-UTR sequences being encoded by exons located further upstream of exon 2. These findings support the concept that different 5'-UTR variants are the result of the different promoters acting in a tissue-specific manner. The association of specific 5'-UTR variants with either GH-R or GH-BP transcripts raises the possibility that the alternative splicing process that generates GH-BP mRNA in the rat might be controlled by the 5'-flanking region regulating the expression of specific leader exons.

Dexamethasone increases growth hormone receptor messenger ribonucleic acid levels in liver and growth plate.

Glucocorticoid inhibits linear growth and renders target tissues, particularly liver and growth plate, insensitive to GH. We hypothesized that glucocorticoid-induced GH insensitivity is due to decreased gene expression of the GH receptor at the messenger RNA (mRNA) level. To test this hypothesis, we treated 4.5-wk-old male rabbits (n = 6-9 per group) with ip dexamethasone or vehicle and measured GH receptor mRNA levels (by RNase protection assay) and serum GH-binding protein levels (by radioimmunoprecipitation assay). Contrary to our hypothesis, dexamethasone administered in growth-suppressing doses did not decrease GH receptor mRNA levels in liver or growth plate. Instead a tissue-specific stimulation of GH receptor mRNA levels was observed. The dose-response relationship of this effect was biphasic, since the lower growth-suppressing dose of dexamethasone (0.1 mg/kg.day) caused the greater increase in GH receptor mRNA levels, whereas the higher growth-suppressing dose (4 mg/kg.day) had less effect. The dexamethasone-induced increase in GH receptor mRNA was observed in growth plate and liver, target tissues important for linear growth, but not in kidney. Serum GH-binding protein levels also showed a stimulatory response to dexamethasone treatment, with a biphasic dose-response relationship. These data suggest that glucocorticoid-induced GH insensitivity cannot be explained by decreased GH receptor mRNA levels. To the contrary, dexamethasone causes a tissue-specific stimulation in GH receptor mRNA levels with a biphasic dose-response relationship.

The effects of estrogen priming and puberty on the growth hormone response to standardized treadmill exercise and arginine-insulin in normal girls and boys.

To determine the effects of puberty and estrogen priming on the GH response to standardized treadmill exercise and arginine-insulin in normal boys and girls, we performed tests in 84 normal children (41 girls and 43 boys) representing all stages of puberty. A subset of the prepubertal children received the tests twice, with or without the administration of ethinyl estradiol (40 micrograms/m2 daily) for 2 days before the tests. The peak GH response to the three tests increased significantly with pubertal stage (r = 0.57; P < 0.0001), but did not differ between boys and girls at the same stage. With advancing puberty, the percentage of normal children who failed to attain a GH level greater than 7 micrograms/L during any of the three tests declined from 61% at pubertal stage 1 to 44% at stage 2, 11% at stage 3, and 0% at stages 4 and 5. Administration of estrogen to the prepubertal subjects raised the normal range for the peak GH response to the three tests from 1.9-20.3 to 7.2-40.5 micrograms/L. We conclude that both puberty and estrogen administration significantly increase the peak GH response to exercise, arginine, or insulin in normal subjects. Moreover, the conventional criterion that the peak GH response to three stimulation tests should exceed 7 micrograms/L was applicable in our study only to subjects who had attained pubertal stage 4 or 5 or who had received estrogen administration.

Growth hormone (GH) stimulates insulin-like growth factor-I (IGF-I) and IGF-binding protein (IGFBP)-2 gene expression in spleens of juvenile rats.

Growth and development of the spleen involves the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis. To evaluate the molecular mechanism of these effects we studied the effect of hypophysectomy (Hx) and GH replacement therapy on the expression of IGF-I, the IGF-I receptor and IGF-binding protein-2 (IGFBP-2) in juvenile rats. Hx resulted in a 30% reduction in body weight. GH replacement therapy for seven days partially prevented these effects. IGF-I mRNA levels were reduced 30% by Hx, IGFBP-2 mRNA levels fell 50% whereas IGF-I receptor mRNA levels were unaffected. GH therapy prevented the reduction in IGF-I and IGFBP-2 mRNA levels. These results suggest that the GH effect on splenic growth and development is via local (paracrine) IGF-I expression, in addition to any effect by circulating (endocrine) IGF-I.

Role of GH and IGF-I in the regulation of IGF-I, IGF-I receptor and IGF binding protein gene expression in the rat spleen.

To characterize the expression of the IGF-I system in the spleen and its role in spleen growth, we have studied the effect of hypophysectomy and the action of either GH or IGF-I treatment on the expression of several components of the IGF system in the rat. Female Sprague-Dawley rats were hypophysectomized (Hx) on postnatal day 50, and five animals each received twice-daily sc injections of saline, bovine GH (bGH; 84 micrograms/animal/day), or recombinant human IGF-I (rhIGF-I; 125 micrograms/animal/day) for 11 days. Compared to sham-operated controls, Hx animals exhibited a reduction in both body (192.6 +/- 5.6 g (mean +/- S.E.M.) vs. 268.6 +/- 6.0 g; P < 0.001) and spleen weights (0.42 +/- 0.03 g vs. 0.84 +/- 0.06 g; P < 0.001). The reduction in body and spleen weights in Hx animals was partially prevented by both bGH and rhIGF-I. Body weights were 234.2 +/- 5.3 g (P < 0.001) after bGH and 213.8 +/- 6.3 g (P < 0.05) after rhIGF-I. Spleen weights were 0.56 +/- 0.048 after bGH P < 0.01 and 0.53 +/- 0.05 g after rhIGF-I (P < 0.05). Serum GH and IGF-I levels were markedly reduced in Hx animals and bGH partially maintained IGF-I levels. Hypophysectomy reduced spleen IGF-I mRNA levels (30.6 +/- 7.5% of control values; P < 0.05) and this reduction was prevented by bGH (96.6 +/- 24.2%; NS) but not by rhIGF-I (39.9 +/- 5.0% NS vs. Hx). There were no changes in GH receptor or IGF-I receptor mRNA levels in Hx or bGH or rhIGF-I-treated animals. When IGF-I binding protein (IGFBP) mRNA levels were studied under these conditions, we found that IGFBP-1 mRNA was not detected in spleen; IGFBP-2 mRNA levels were reduced in Hx rats (67.9 +/- 7.4% of control values, P < 0.05) and bGH treatment prevented this reduction (95.5 +/- 12.2%, NS). IGFBP-3 mRNA levels were not affected by hypophysectomy or by bGH treatment, but were reduced in rhIGF-treated rats (69.6 +/- 3.0%, P < 0.05). On the other hand, IGFBP-4 mRNA levels were increased in Hx rats (136.4 +/- 15.9% of control values, P < 0.05) and bGH treatment prevented this increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol inhibits growth hormone receptor gene expression in rabbit liver.

We studied the ontogeny of GH receptor mRNA levels and the effect of exogenous estradiol administration on GH receptor mRNA levels in rabbit liver. A solution hybridization-RNase protection assay revealed a predominant 370-base long protected band corresponding to the mRNA encoding the transmembrane GH receptor, and a 241-base long protected band, representing about 9.0%, with the predicted size for the truncated form of the GH receptor. To study the developmental profile of GH receptor expression, we studied 12 female rabbits, at ages 1, 3, 5 and 7 months. Maximal GH receptor mRNA levels were observed in 3-month-old animals and decreased in 7-month-old animals. To investigate the effect of estradiol, 8-week-old immature female rabbits were randomly divided into five groups, and received subcutaneous pellets containing either placebo or estradiol at doses of 0.1, 0.5, 1.5 and 5.0 mg for 3 weeks. Exogenous administration of estradiol, at doses that resulted in physiological circulating levels, induced a reduction in GH receptor expression, measured both by GH binding (36 and 46%), and GH receptor mRNA levels (38 and 87%), in animals receiving pellets containing 1.5 and 5.0 mg of estradiol, respectively. We conclude that estradiol decreases GH receptor expression in rabbit liver. The results of our study suggest that there is an inverse relationship between circulating estrogen concentrations and liver GH receptor expression.

Growth hormone (GH) stimulates insulin-like growth factor-I (IGF-I) and IGF-I-binding protein-3, but not GH receptor gene expression in livers of juvenile rats.

In the adult rat, expression of the liver GH receptor, insulin-like growth factor-I (IGF-I), and IGF-I-binding protein-3 (IGFBP-3) genes has been shown to be under GH control. Additionally, hypophysectomy and GH treatment have a differential effect on the relative abundance of liver IGF-I mRNA variants in adult rats. To further elucidate the time of appearance and the extent of GH control of liver GH receptor, IGF-I, and IGFBP-3 gene expression, we studied the effect of hypophysectomy and GH and IGF-I treatment in juvenile rats. Male Wistar rats were hypophysectomized (Hx) on postnatal day 26 and received twice daily sc injections of saline, recombinant human GH (2.5 U/kg.day), or recombinant human IGF-I (500 micrograms/kg.day) for 7 days. Sham-operated rats received the same treatment. Hx animals also received T4 (20 micrograms/kg.day). In Hx animals, there was a significant reduction in body weight (69.8 +/- 6.6 vs. 100.4 +/- 5.4 g; P < 0.001). GH, but not IGF-I, treatment increased body weight (79.6 +/- 9.6 g after GH vs. 69.8 +/- 6.6 g before GH; P < 0.05). GH treatment partially maintained liver, kidney, and lung weights in Hx animals and increased them in intact animals, whereas IGF-I treatment did so only in the lungs of intact and Hx animals. Serum GH and IGF-I levels were markedly reduced in Hx animals compared with those in intact controls, and GH treatment maintained, albeit partially, circulating IGF-I levels compared with those in saline-treated Hx animals. IGF-I mRNA levels were markedly reduced in Hx liver (25.0 +/- 5.4%; P < 0.001 compared with intact controls). GH treatment for 7 days increased IGF-I mRNA levels by 4.8-fold over the levels in 9-day Hx animals and increased IGF-I mRNA levels by 2.2-fold in control rats. Hypophysectomy decreased exon 2-containing transcripts by 7.0-fold and exon 1-containing transcripts by 4.1-fold. GH treatment, however, affected both exon 1- and exon 2-containing transcripts similarly. Hepatic IGFBP-3 mRNA levels were reduced in Hx (53.2 +/- 1.8%; P < 0.01 compared with intact controls) and IGF-treated Hx animals, but were not decreased in Hx GH-treated animals (100.6 +/- 9.5). No changes in GH receptor or GH-binding protein mRNA levels were caused by Hx, GH, or IGF-I treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterologous humoral immune response in patients treated with human growth hormone from different sources.

The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [125I]hGH or [125I]bGH as tracers. Fifty-seven hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[125]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such a fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response.

High serum sex hormone-binding globulin (SHBG) and low serum non-SHBG-bound testosterone in boys with idiopathic hypopituitarism: effect of recombinant human growth hormone treatment.

We measured serum sex hormone-binding globulin (SHBG), total testosterone (T), non-SHBG-bound T, albumin-bound T, free T, and SHBG-bound T in 19 prepubertal boys with hypopituitarism. Serum SHBG decreased with age with a slope similar to that in 91 normal prepubertal boys at higher level, and therefore, it reached similar values at a later age. Serum SHBG was significantly higher in hypopituitary prepubertal boys [mean, 123 +/- 12 (+/- SE) nmol/L] than in normal prepubertal boys (76 +/- 4; P less than 0.001) despite the fact that their mean age was also higher (10.0 +/- 4 vs. 7.1 +/- 4.1 yr; P less than 0.001). In 4 boys with isolated hypogonadotropic hypogonadism (Kallman's syndrome), aged 15.6 +/- 1.5 yr, serum SHBG was 21 +/- 14 nmol/L, a value below the 95% confidence limit of the regression line in GH-deficient boys. The affinity constants of association of the SHBG-DHT complex were similar in hypopituitary and normal boys. Eleven of the 19 hypopituitary boys (mean chronological age, 8.3 +/- 2.5 yr; mean bone age, 4.1 +/- 2.1 yr) were treated with recombinant hGH (0.5 U/kg BW.week) for 1 yr. Their mean serum SHBG level before treatment was 154 +/- 14 nmol/L, and it decreased gradually to 106 +/- 5 nmol/L (P less than 0.01) after 12 months of treatment. The tendency toward normalization of serum SHBG during treatment suggested that GH deficiency was responsible for the high serum SHBG levels. Serum SHBG correlated negatively with age in both treated hypopituitary and normal boys, but the slope of the regression line was significantly steeper in treated hypopituitary boys (P less than 0.01). On the other hand, the mean serum non-SHBG-bound T level was 0.10 +/- 0.02 (+/- SE) nmol/L in hypopituitary boys, significantly lower than that in normal boys (0.21 +/- 0.02 nmol/L; P less than 0.02). Since serum total T concentrations were similar in the two groups, the higher serum SHBG concentration resulted in lower serum bioavailable T levels in the hypopituitary boys. These changes might explain the poor response to T treatment reported in GH-deficient patients. The lower serum non-SHBG-bound T concentrations in the GH-deficient boys suggest there may be delayed exposure of central nervous system structures to increased levels of sex hormones, which, in turn, may slow body maturation. This mechanism might play a role in the delay of puberty that occurs in patients with isolated GH deficiency.