Growth hormone-binding protein in normal and pathologic gestation: Correlations with maternal diabetes and fetal growth

To date, measurements of GH-binding protein (GHBP) during human pregnancy have been carried out using;assays susceptible to interference by the elevated levels of human placental GH typical of late gestation. We recruited a large cohort of pregnant women (n = 140) for serial measurements of GHBP and used the ligand immunofunctional assay for GHBP. For normal gravidas, GHBP levels fell throughout gestation. Mean levels were 1.07 nmol/L (SE = 0.18) in the first trimester, 0.90 nmol/L (SE = 0.08) at 18-20 weeks, 0.73 nmol/L (SE = 0.05) at 28-30 weeks, and 0.62 nmol/L (SE = 0.06) at 36-38 weeks. GHBP levels in the first trimester correlated significantly with maternal body mass index (r = 0.58; P < 0.01). GHBP levels in pregnancies complicated by noninsulin-dependent diabetes mellitus (NIDDM) were substantially elevated at all gestational ages. The mean value in the first quarter (2.29 nmol/L) was more than double the normal mean (P < 0.01). In contrast, patients with insulin-dependent diabetes mellitus (IDDM) showed reduced GHBP concentrations at 36-38 weeks. The correlation between body mass index and GHBP is consistent with a metabolic role for GHBP during pregnancy, as is the dramatic elevation in GHBP observed in cases of NIDDM. At 36 weeks gestation, GHBP was significantly elevated (P < 0.01) in those women whose neonates had low birth weight (

Analysis of Craniofacial and Extremity Growth in Patients with Growth Hormone Deficiency during Growth Hormone Therapy

Background/Aims: There are many controversies regarding side effects on craniofacial and extremity growth due to growth hormone ( GH) treatment. Our aim was to estimate GH action on craniofacial development and extremity growth in GH-deficient patients. Methods: Twenty patients with GH deficiency with a chronological age ranging from 4.6 to 24.3 years (bone age from 1.5 to 13 years) were divided in 2 groups: group 1 (n = 6), naive to GH treatment, and group 2 (n = 14), ongoing GH treatment for 2-11 years. GH doses (0.1 -0.15 U/kg/day) were adjusted to maintain insulin-like growth factor 1 and insulin-like growth factor binding protein 3 levels within the normal range. Anthropometric measurements, cephalometric analyses and facial photographs to verify profile and harmony were performed annually for at least 3 years. Results: Two patients with a disharmonious profile due to mandibular growth attained harmony, and none of them developed facial disharmony. Increased hand or foot size (>P97) was observed in 2 female patients and in 4 patients (1 female), respectively, both not correlated with GH treatment duration and increased levels of insulin-like growth factor 1. Conclusions: GH treatment with standard doses in GH-deficient patients can improve the facial profile in retrognathic patients and does not lead to facial disharmony although extremity growth, mainly involving the feet, can occur. Copyright (C) 2009 S. Karger AG, Basel

Consequences of lifetime isolated growth hormone (GH) deficiency and effects of short-term GH treatment on bone in adults with a mutation in the GHRH-receptor gene

Growth hormone (GH) influences bone mass maintenance. However, the consequences of lifetime isolated GH deficiency (IGHD) on bone are not well established. We assessed the bone status and the effect of 6 months of GH replacement in GH-naive adults with IGHD due to a homozygous mutation of the GH-releasing hormone (GHRH)-receptor gene (GHRHR). We studied 20 individuals (10 men) with IGHD at baseline, after 6 months of depot GH treatment, and 6 and 12 months after discontinuation of GH. Quantitative ultrasound (QUS) of the heel was performed and serum osteocalcin (OC) and C-terminal cross-linking telopeptide of type I collagen (ICTP) were measured. QUS was also performed at baseline and 12 months later in a group of 20 normal control individuals (CO), who did not receive GH treatment. At baseline, the IGHD group had a lower T-score on QUS than CO (-1.15 +/- 0.9 vs. -0.07 +/- 0.9, P < 0.001). GH treatment improved this parameter, with improvement persisting for 12 months post-treatment (T-score for IGHD = -0.59 +/- 0.9, P < 0.05). GH also caused an increase in serum OC (baseline vs. pGH, P < 0.001) and ICTP (baseline vs. pGH, P < 0.01). The increase in OC was more marked during treatment and its reduction was slower after GH discontinuation than in ICTP. These data suggest that lifetime severe IGHD is associated with significant reduction in QUS parameters, which are partially reversed by short-term depot GH treatment. The treatment induces a biochemical pattern of bone anabolism that persists for at least 6 months after treatment discontinuation.

Cholecystokinin and hypothalamic corticotrophin-releasing factor participate in endotoxin-induced hypophagia

Cholecystokinin (CCK) provides a meal-related signal that activates brainstem neurons, which have reciprocal interconnections with the hypothalamic paraventricular nucleus. Neurons that express corticotrophin-releasing factor (CRF) in the hypothalamus possess anorexigenic effects and are activated during endotoxaemia. This study investigated the effects of CCK(1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia and hypothalamic CRF neuronal activation. Male Wistar rats were pretreated with a specific CCK(1) receptor antagonist (devazepide; 1 mg kg(-1); I.P.) or vehicle; 30 min later they received LPS (100 mu g kg(-1); I.P.) or saline injection. Food intake, corticosterone responses and Fos-CRF and Fos-alpha-melanocyte-stimulating hormone (alpha-MSH) immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase immunoreactivity in the nucleus of the solitary tract (NTS) were evaluated. In comparison with saline treatment, LPS administration decreased food intake and increased plasma corticosterone levels, as well as the number of Fos-CRF and Fos-tyrosine hydroxylase double-labelled neurons in vehicle-pretreated rats; no change in Fos-alpha-MSH immunoreactivity was observed after LPS injection. In saline-treated animals, devazepide pretreatment increased food intake, but it did not modify other parameters compared with vehicle-pretreated rats. Devazepide pretreatment partly reversed LPS-induced hypophagia and Fos-CRF and brainstem neuronal activation. Devazepide did not modify the corticosterone and Fos-alpha-MSH responses in rats treated with LPS. In conclusion, the present data suggest that LPS-induced hypophagia is mediated at least in part by CCK effects, via CCK(1) receptor, on NTS and hypothalamic CRF neurons.

Prostaglandin mediates endotoxaemia-induced hypophagia by activation of pro-opiomelanocortin and corticotrophin-releasing factor neurons in rats

Corticotrophin-releasing factor (CRF) and alpha-melanocyte-stimulating hormone (alpha-MSH), both of which are synthesized by hypothalamic neurons, play an essential role in the control of energy homeostasis. Neuroendocrine and behavioural responses induced by lipopolyssacharide (LPS) have been shown to involve prostaglandin-mediated pathways. This study investigated the effects of prostaglandin on CRF and alpha-MSH neuronal activities in LPS-induced anorexia. Male Wistar rats were pretreated with indomethacin (10 mg kg(-1); i.p.) or vehicle; 15 min later they received LPS (500 mu g kg(-1); i.p.) or saline injection. Food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the paraventricular and arcuate nuclei, respectively, were evaluated. In comparison with saline treatment, LPS administration induced lower food intake and increased plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF and Fos-alpha-MSH double-labelled neurons in vehicle-pretreated rats. In contrast, indomethacin treatment partly reversed the hypophagic effect, blunted the hormonal increase and blocked the Fos-CRF and Fos-alpha-MSH hypothalamic double labelling increase in response to the LPS stimulus. These data demonstrate that the activation of pro-opiomelanocortin and CRF hypothalamic neurons following LPS administration is at least partly mediated by the prostaglandin pathway and is likely to be involved in the modulation of feeding behaviour during endotoxaemia.

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POW mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 mu g/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POW neurons in the hypothalamus and an increased mRNA expression of these neuropeptides. (C) 2008 Elsevier Inc. All rights reserved.

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.

Growth hormone therapy in Prader-Willi Syndrome

Prader-Willi syndrome (PWS) was originally described less than 50 y ago,1 although reference to children with characteristics of the syndrome are to be found in other literature previous to this.2 Until relatively recently the diagnosis was made upon the clinical features as outlined by Holm,3 which include severe muscular hypotonia in the neonatal period leading to feeding difficulties and undernutrition, hypogonadism and later hyperphagia and obesity. Latterly the syndrome has been identified as being associated with an interstitial deletion of the q11-13 region on chromosome 15.4 In the majority of cases the deletion is in the paternally derived chromosome. In the remainder of cases there seems to be a failure to inherit the entire paternal chromosome and as a consequence both the chromosomes inherited are maternal, thus leading to maternal disomy.

Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimers

GH actions are dependent on receptor dimerization. The GH receptor antagonist, B2036-PEG, has been developed for treating acromegaly. B2036 has mutations in site 1 to enhance receptor binding and in site 2 to block receptor dimerization. Pegylation (B2036-PEG) increases half-life and lowers immunogenicity, but high concentrations are required to control insulin-like growth factor-I levels. We examined antagonist structure and function and the impact of pegylation on biological efficacy. Unpegylated B2036 had a 4.5-fold greater affinity for GH binding protein (GHBP) than GH but similar affinity for membrane receptor. Pegylation substantially reduced membrane binding affinity and receptor antagonism, as assessed by a transcription assay, by 39- and 20-fold, respectively. GHBP reduced antagonist activity of unpegylated B2036 but did not effect antagonism by B2036-PEG. B2036 down-regulated receptors, and membrane binding sites doubled in the presence of dimerization-blocking antibodies, suggesting that B2036 binds to a receptor dimer. It is concluded that the high concentration requirement of B2036-PEG for clinical efficacy relates to pegylation, which decreases binding to membrane receptor but has the advantages of reduced clearance, immunogenicity, and interactions with GHBP. Our studies suggest that B2036 binds to a receptor dimer and induces internalization but not signaling.

Changes in non-22-kilodalton (kDa) isoforms of growth hormone (GH) after administration of 22-kDa recombinant human GH in trained adult males

GH is being used by elite athletes to enhance sporting performance. To examine the hypothesis that exogenous 22-kDa recombinant human GH (rhGH) administration could be detected through suppression of non-22-kDa isoforms of GH, we studied seventeen aerobically trained males (age, 26.9 +/- 1.5 yr) randomized to rhGH or placebo treatment (0.15 IU/kg/day for 1 week). Subjects were studied at rest and in response to exercise (cycle-ergometry at 65% of maximal work capacity for 20 min). Serum was assayed for total GH (Pharmacia IRMA and pituitary GH), 22-kDa GH (2 different 2-site monoclonal immunoassays), non-22-kDa GH (22-kDa GH-exclusion assay), 20-kDa GH, and immunofunctional GH. In the study, 3 h after the last dose of rhGH, total and 22-kDa GH concentrations were elevated, reflecting exogenous 22-kDa GH. Non-22-kDa and 20-kDa GH levels were suppressed. Regression of non-22-kDa or 20-kDa GH against total or 22-kDa GH produced clear separation of treatment groups. In identical exercise studies repeated between 24 and 96 h after cessation of treatment, the magnitude of the responses of all GH isoforms was suppressed (P < 0.01), but the relative proportions were similar to those before treatment. We conclude: 1) supraphysiological doses of rhGH in trained adult males suppressed exercise-stimulated endogenous circulating isoforms of GH for up to 4 days; 2) the dearest separation of treatment groups required the simultaneous presence of high exogenous 22-kDa GH and suppressed 20-kDa or non-22-kDa GH concentrations; and 3) these methods may prove useful in detecting rhGH abuse in athletes.

The response of molecular isoforms of growth hormone to acute exercise in trained adult males

Circulating GH consists of multiple molecular isoforms, all derived from the one gene in nonpregnant humans. To assess the effect of a potent stimulus to pituitary secretion on GH isoforms, we studied 17 aerobically trained males (age, 26.9 +/- 1.5 yr) in a randomized, repeat measures study of rest vs. exercise. Exercise consisted of continuous cycle ergometry at approximately 80% of predetermined maximal oxygen uptake for 20 min. Serum was assayed for total, pituitary, 22-kDa, recombinant, non-22-kDa, 20-kDa, and immunofunctional GH. All isoforms increased during, peaked at the end, and declined after exercise. At peak exercise, 22-kDa GH was the predominant isoform. After exercise, the ratios of non-22 kDa/total GH and 20-kDa GH/total GH increased and those of recombinant/pituitary GH decreased. The disappearance half-times for pituitary GH and 20-kDa GH were significantly longer than those for all other isoforms. We conclude that 1) all molecular isoforms of GH measured increased with and peaked at the end of acute exercise, with 22-kBa GH constituting the major isoform in serum during exercise; and 2) the proportion of non-22-kDa isoforms increased after exercise due in part to slower disappearance rates of 20-kDa and perhaps other non-22-kDa GH isoforms. It remains to be determined whether the various biological actions of different GH isoforms impact on postexercise homeostasis.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.

Growth hormone is permissive for skeletal adaptation to mechanical loading

The Lewis dwarf (DW) rat was used as a model to test the hypothesis that growth hormone (GH) is permissive for new bone formation induced by mechanical loading in vivo. Adult female Lewis DW rats aged 6.2 +/- 0.1 months (187 +/- 18 g) were allocated to four vehicle groups (DW), four GH treatment groups at 32.5 mug/100 g body mass (DWGH1), and four GH treatment groups at 65 mug/100 g (DWGH2). Saline vehicle or GH was injected intraperitoneally (ip) at 6:30 p.m. and 6:30 a.m. before mechanical loading of tibias at 7:30 a.m. A single period of 300 cycles of four-point bending was applied to right tibias at 2.0 Hz, and magnitudes of 24, 29, 38, or 48N were applied. Separate strain gauge analyses in 5 DW rats validated the selection of loading magnitudes. After loading, double-label histomorphometry was used to assess bone formation at the periosteal surface (Ps.S) and endocortical surface (Ec.S) of tibias. Comparing left (unloaded) tibias among groups, GH treatment had no effect on bone formation. Bone formation in tibias in DW rats was insensitive to mechanical loading. At the Ec.S, mechanically induced lamellar bone formation increased in the DWGH2 group loaded at 48N (p < 0.05), and no significant increases in bone formation were observed among other groups. The percentage of tibias expressing woven bone formation (Wo.B) at the Ps.S was significantly greater in the DWGH groups compared with controls (p < 0.05). We concluded that GH influences loading-related bone formation in a permissive manner and modulates the responsiveness of bone tissue to mechanical stimuli by changing thresholds for bone formation.

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid D-Phe in aSvg-30 [D-Phe11,His12] Svg((11-40)) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl) benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[ His12] Svg was tested for its ability to displace [I-125-Tyr0] oCRF or [I-125-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 ( rCRF(1)) or mouse CRF receptor, type 2beta (mCRF(2beta)). Furthermore, the ability of ATB- [His12] Svg((12-40)) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF(1) (HEK-rCRF(1) cells) or mCRF(2beta) (HEK-mCRF(2beta) cells) was determined. Unlike astressin and photo astressin, ATB- [His12]Svg((12-40)) showed high selective binding to mCRF(2beta) (K-i = 3.1 +/- 0.2 nM) but not the rCRF(1) receptor (K-i = 142. 5 +/- 22.3 nM) and decreased Svg-stimulated cAMP activity in mCRF(2beta)-expressing cells in a similar fashion as aSvg-30. A66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB- [His12]Svg((12-40)) was covalently linked to mCRF(2beta) receptor. The specificity of the photoactivatable I-125-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy

Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PCH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20% with glucose loading. Cot-relation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not Suppressed by oral glucose loading in non-diabetic mothers.