Effect of zinc binding residues in growth hormone (GH) and altered intracellular zinc content on regulated GH secretion.

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn(2+) binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn(2+) from culture medium using N,N,N',N'-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn(2+) chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn(2+) to culture medium increased intracellular free Zn(2+) levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn(2+) levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn(2+) as well as altering intracellular free Zn(2+) content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.

Short stature caused by a biologically inactive mutant growth hormone (GH-C53S).

Human GH has two disulfide bridges linking Cys-53 to Cys-165 and Cys-182 to Cys-189. Although absence of the first disulfide bridge has been shown to affect the bioactivity of GH in transgenic mice, little is known of the importance of this bridge in mediating the GH/GH-receptor (GHR) interaction in humans. However, we have identified a missense mutation (G705C) in the GH1 gene of a Serbian patient. This mutation was found in the homozygous state and leads to the absence of the disulfide bridge Cys-53 to Cys-165. To study the impact of this mutation in vitro, GHR binding and Janus kinase (Jak)2/signal transducer and activator of transcription (Stat)5 activation experiments were performed, in which it was observed that at physiological concentrations (3-50 ng/ml) both GHR binding and Jak2/Stat5 signaling pathway activation were significantly reduced in the mutant GH-C53S, compared with wild-type (wt)-GH. Higher concentrations (400 ng/ml) were required for this mutant to elicit responses similar to wt-GH. These results demonstrate that the absence of the disulfide bridge Cys-53 to Cys-165 affects the binding affinity of GH for the GHR and subsequently the potency of GH to activate the Jak2/Stat5 signaling pathway. In conclusion, we have demonstrated that GH-C53S is a bioinactive GH at the physiological range and that the disulfide bridge Cys-53 to Cys-163 is required for mediating the biological effects of GH.

"Hot spot" in the PROP1 gene responsible for combined pituitary hormone deficiency.

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). Although pit-1 was 1 of the first factors identified as a cause of CPHD in mice, many other homeodomain and transcription factors have been characterized as being involved in different developmental stages of pituitary gland development, such as prophet of pit-1 (prop-1), P-Lim, ETS-1, and Brn 4. The aims of the present study were first to screen families and patients suffering from different forms of CPHD for PROP1 gene alterations, and second to define possible hot spots and the frequency of the different gene alterations found. Of 73 subjects (36 families) analyzed, we found 35 patients, belonging to 18 unrelated families, with CPHD caused by a PROP1 gene defect. The PROP1 gene alterations included 3 missense mutations, 2 frameshift mutations, and 1 splice site mutation. The 2 reported frameshift mutations could be caused by any 2-bp GA or AG deletion at either the 148-GGA-GGG-153 or 295-CGA-GAG-AGT-303 position. As any combination of a GA or AG deletion yields the same sequencing data, the frameshift mutations were called 149delGA and 296delGA, respectively. All but 1 mutation were located in the PROP1 gene encoding the homeodomain. Importantly, 3 tandem repeats of the dinucleotides GA at location 296-302 in the PROP1 gene represent a hot spot for CPHD. In conclusion, the PROP1 gene seems to be a major candidate gene for CPHD; however, further studies are needed to evaluate other genetic defects involved in pituitary development.

Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43

Butyrate is a short-chain fatty acid (SCFA) closely related to the ketone body ß-hydroxybutyrate (BHB), which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH) rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR), GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH)-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

Alteration of ZnT5-mediated zinc import into the early secretory pathway affects the secretion of growth hormone from rat pituitary cells

BACKGROUND Aggregation of growth hormone (GH) required for its proper storage in granules is facilitated by zinc (Zn(2+)) transported by specific zinc transporters in and out of the regulated secretory pathway. Slc30a5 (ZnT5) was reported to have the highest gene expression among all zinc transporters in primary mouse pituitary cells while ZnT5-null mice presented with abnormal bone development and impaired growth compared to wild-type counterparts. METHODS In vitro studies performed in GH3 cells, a rat pituitary cell line that endogenously produces rat GH (rGH), included analysis of: cytoplasmic Zn(2+) pool changes after altering rSlc30a5 expression (luciferase assay), rZnT5 association with different compartments of the regulated secretory pathway (confocal microscopy), and the rGH secretion after rSlc30a5 knock-down (Western blot). RESULTS Confocal microscopy demonstrated high co-localization of rZnT5 with ER and Golgi (early secretory pathway) while siRNA-mediated knock-down of rSlc30a5 gene expression led to a significant reduction in rGH secretion. Furthermore, altered expression of rSlc30a5 (knock-down/overexpression) evoked changes in the cytoplasmic Zn(2+) pool indicating its important role in mediating Zn(2+) influx into intracellular compartments of the regulated secretory pathway. CONCLUSION Taken together, these results suggest that ZnT5 might play an important role in regulated GH secretion that is much greater than previously anticipated.

A 2-year multicentre, open-label, randomized, controlled study of growth hormone (Genotropin(®) ) treatment in very young children born small for gestational age: Early Growth and Neurodevelopment (EGN) Study

OBJECTIVE In Europe, growth hormone (GH) treatment for children born small for gestational age (SGA) can only be initiated after 4 years of age. However, younger age at treatment initiation is a predictor of favourable response. To assess the effect of GH treatment on early growth and cognitive functioning in very young (<30 months), short-stature children born SGA. DESIGN A 2-year, randomized controlled, multicentre study (NCT00627523; EGN study), in which patients received either GH treatment or no treatment for 24 months. PATIENTS Children aged 19-29 months diagnosed as SGA at birth, and for whom sufficient early growth data were available, were eligible. Patients were randomized (1:1) to GH treatment (Genotropin(®) , Pfizer Inc.) at a dose of 0·035 mg/kg/day by subcutaneous injection, or no treatment. MEASUREMENTS The primary objective was to assess the change from baseline in height standard deviation score (SDS) after 24 months of GH treatment. RESULTS Change from baseline in height SDS was significantly greater in the GH treatment vs control group at both month 12 (1·03 vs 0·14) and month 24 (1·63 vs 0·43; both P < 0·001). Growth velocity SDS was significantly higher in the GH treatment vs control group at 12 months (P < 0·001), but not at 24 months. There was no significant difference in mental or psychomotor development indices between the two groups. CONCLUSIONS GH treatment for 24 months in very young short-stature children born SGA resulted in a significant increase in height SDS compared with no treatment.

Genetic evaluation of short stature

After a proper medical history, growth analysis and physical examination of a short child, followed by radiological and laboratory screening, the clinician may decide to perform genetic testing. We propose several clinical algorithms that can be used to establish the diagnosis. GH1 and GHRHR should be tested in children with severe isolated growth hormone deficiency and a positive family history. A multiple pituitary dysfunction can be caused by defects in several genes, of which PROP1 and POU1F1 are most common. GH resistance can be caused by genetic defects in GHR, STAT5B, IGF1, IGFALS, which all have their specific clinical and biochemical characteristics. IGF-I resistance is seen in heterozygous defects of the IGF1R. If besides short stature additional abnormalities are present, these should be matched with known dysmorphic syndromes. If no obvious candidate gene can be determined, a whole genome approach can be taken to check for deletions, duplications and/or uniparental disomies.

Impact of del32-71-GH (exon 3 skipped GH) on intracellular GH distribution, secretion and cell viability: a quantitative confocal microscopy analysis

BACKGROUND: Familial isolated growth hormone deficiency (IGHD) is a disorder with about 5-30% of patients having affected relatives. Among those familial types, IGHD type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32-71-GH, a GH peptide which cannot fold properly. The mechanism by which this mutant GH may alter the controlled secretory pathway and therefore suppress the secretion of the normal 22-kDa GH product of the normal allele is not known in detail. Previous studies have shown variance depending on cell type, transfection technique used, as well as on the method of analysis performed. AIM: The aim of our study was to analyse and compare the subcellular distribution/localization of del32-71-GH or wild-type (wt)-GH (22-kDa GH), each stably transfected into AtT-20, a mouse pituitary cell line endogenously producing ACTH, employed as the internal control for secretion assessment. METHODS: Colocalization of wt- and del32-71 mutant GH form was studied by quantitative confocal microscopy analysis. Using the immunofluorescent technique, cells were double stained for GH plus one of the following organelles: endoplasmic reticulum (ER anti-Grp94), Golgi (anti-betaCOP) or secretory granules (anti-Rab3a). In addition, GH secretion and cell viability were analysed in detail. RESULTS/CONCLUSIONS: Our results show that in AtT-20 neuroendocrine cells, in comparison to the wt-GH, the del32-71-GH has a major impact on the secretory pathway not only affecting GH but also other peptides such as ACTH. The del32-71-GH is still present at the secretory vesicles' level, albeit in reduced quantity when compared to wt-GH but, importantly, was secretion-deficient. Furthermore, while focusing on cell viability an additional finding presented that the various splice site mutations, even though leading eventually to the same end product, namely del32-71-GH, have different and specific consequences on cell viability and proliferation rate.

Patients with biallelic mutations in the chloride channel gene CLCNKB: long-term management and outcome

BACKGROUND: Little information on the management and long-term follow-up of patients with biallelic mutations in the chloride channel gene CLCNKB is available. METHODS: Long-term follow-up was evaluated from 5.0 to 24 years (median, 14 years) after diagnosis in 13 patients with homozygous (n = 10) or compound heterozygous (n = 3) mutations. RESULTS: Medical treatment at last follow-up control included supplementation with potassium in 12 patients and sodium in 2 patients and medical treatment with indomethacin in 9 patients. At the end of follow-up, body height was 2.0 standard deviation score or less in 6 patients; 2 of these patients had growth hormone deficiency. Body weight (growth retardation is common and sometimes related to growth hormone deficiency in these patients.

Suprasellar germinomas in childhood and adolescence: diagnostic pitfalls

The clinical and neuro-endocrine data of seven young male patients with suprasellar germinomas seen between 1984 and 1992 are reported. The most common initial symptom was 'idiopathic' central diabetes insipidus (DI), which occurred in all seven patients. The time interval between the appearance of this first clinical sign and the definitive diagnosis of a suprasellar germinoma ranged from 3 to 66 months. Raised prolactin levels and growth hormone deficiency were indicators of a process located in the hypothalamic-pituitary region. An increased beta-HCG level in the serum or the CSF confirmed the diagnostic suspicion of a germinoma and was helpful as a tumor marker in follow-up. Neuro-radiologic studies (CT or MRI) were also disappointing in the early stage when patients presented only with DI. Later on, as patients developed additional symptoms or signs related to the tumor, imaging studies were positive. Given the variable rate of tumor progression, the nonspecific early signs of hypothalamic-pituitary dysfunction (DI) as well as the often negative early imaging studies, the diagnosis of suprasellar germinoma is difficult but should always be considered in the presence of so-called 'idiopathic' central DI. Repeated brain MRIs are mandatory in young patients with idiopathic DI in order not to miss an underlying suprasellar germinoma.

Transient Neonatal Zinc Deficiency Caused by a Heterozygous G87R Mutation in the Zinc Transporter ZnT-2 (SLC30A2) Gene in the Mother Highlighting the Importance of Zn (2+) for Normal Growth and Development

Suboptimal dietary zinc (Zn(2+)) intake is increasingly appreciated as an important public health issue. Zn(2+) is an essential mineral, and infants are particularly vulnerable to Zn(2+) deficiency, as they require large amounts of Zn(2+) for their normal growth and development. Although term infants are born with an important hepatic Zn(2+) storage, adequate Zn(2+) nutrition of infants mostly depends on breast milk or formula feeding, which contains an adequate amount of Zn(2+) to meet the infants' requirements. An exclusively breast-fed 6 months old infant suffering from Zn(2+) deficiency caused by an autosomal dominant negative G87R mutation in the Slc30a2 gene (encoding for the zinc transporter 2 (ZnT-2)) in the mother is reported. More than 20 zinc transporters characterized up to date, classified into two families (Slc30a/ZnT and Slc39a/Zip), reflect the complexity and importance of maintaining cellular Zn(2+) homeostasis and dynamics. The role of ZnTs is to reduce intracellular Zn(2+) by transporting it from the cytoplasm into various intracellular organelles and by moving Zn(2+) into extracellular space. Zips increase intracellular Zn(2+) by transporting it in the opposite direction. Thus the coordinated action of both is essential for the maintenance of Zn(2+) homeostasis in the cytoplasm, and accumulating evidence suggests that this is also true for the secretory pathway of growth hormone.

A novel GH-1 gene mutation (GH-P59L) causes partial GH deficiency type II combined with bioinactive GH syndrome

Despite the differences in the main characteristics between the autosomal dominant form of GH deficiency (IGHD II) and the bioinactive GH syndrome, a common feature of both is their impact on linear growth leading to short stature in all affected patients.

Effect of GH on human skeletal muscle lipid metabolism in GH deficiency

Adult-onset growth hormone (GH) deficiency (GHD) is associated with insulin resistance and decreased exercise capacity. Intramyocellular lipids (IMCL) depend on training status, diet, and insulin sensitivity. Using magnetic resonance spectroscopy, we studied IMCL content following physical activity (IMCL-depleted) and high-fat diet (IMCL-repleted) in 15 patients with GHD before and after 4 mo of GH replacement therapy (GHRT) and in 11 healthy control subjects. Measurements of insulin resistance and exercise capacity were performed and skeletal muscle biopsies were carried out to assess expression of mRNA of key enzymes involved in skeletal muscle lipid metabolism by real-time PCR and ultrastructure by electron microscopy. Compared with control subjects, patients with GHD showed significantly higher difference between IMCL-depleted and IMCL-repleted. GHRT resulted in an increase in skeletal muscle mRNA expression of IGF-I, hormone-sensitive lipase, and a tendency for an increase in fatty acid binding protein-3. Electron microscopy examination did not reveal significant differences after GHRT. In conclusion, variation of IMCL may be increased in patients with GHD compared with healthy control subjects. Qualitative changes within the skeletal muscle (i.e., an increase in free fatty acids availability from systemic and/or local sources) may contribute to the increase in insulin resistance and possibly to the improvement of exercise capacity after GHRT. The upregulation of IGF-I mRNA suggests a paracrine/autocrine role of IGF-I on skeletal muscle.

Genetics of GHRH, GHRH-receptor, GH and GH-receptor: its impact on pharmacogenetics

When a child is not following the normal, predicted growth curve, an evaluation for underlying illnesses and central nervous system abnormalities is required and, appropriate consideration should be given to genetic defects causing GH deficiency (GHD). Because Insulin-like-Growth Factor-I (IGF-I) plays a pivotal role, GHD could also be considered as a form of IGF-I deficiency (IGFD). Although IGFD can develop at any level of the GHRH-GH-IGF axis, a differentiation should be made between GHD (absent to low GH in circulation) and IGFD (normal to high GH in circulation). The main focus of this review is on the GH-gene, the various gene alterations and their possible impact on the pituitary gland. However, although transcription factors regulating the pituitary gland development may cause multiple pituitary hormone deficiency they may present initially as GHD. These defects are discussed in various different chapters within this book, whereas, the impact of alterations of the GHRH-, GHRH-receptor- --as well as the GH-receptor (GHR) gene--will be discussed here.

Endocrine changes and liver mRNA abundance of somatotropic axis and insulin system constituents during negative energy balance at different stages of lactation in dairy cows

The liver has an important role in metabolic regulation and control of the somatotropic axis to adapt successfully to physiological and environmental changes in dairy cows. The aim of this study was to investigate the adaptation to negative energy balance (NEB) at parturition and to a deliberately induced NEB by feed restriction at 100 days in milk. The hepatic gene expression and the endocrine system of the somatotropic axis and related parameters were compared between the early and late NEB period. Fifty multiparous cows were subjected to 3 periods (1=early lactation up to 12 wk postpartum, 2=feed restriction for 3 wk beginning at around 100 days in milk with a feed-restricted and a control group, and 3=subsequent realimentation period for the feed-restricted group for 8 wk). In period 1, plasma growth hormone reached a maximum in early lactation, whereas insulin-like growth factor-I (IGF-I), leptin, the thyroid hormones, insulin, and the revised quantitative insulin sensitivity check index increased gradually after a nadir in early lactation. Three days after parturition, hepatic mRNA abundance of growth hormone receptor 1A, IGF-I, IGF-I receptor and IGF-binding protein-3 (IGFBP-3) were decreased, whereas mRNA of IGFBP-1 and -2 and insulin receptor were upregulated as compared with wk 3 antepartum. During period 2, feed-restricted cows showed decreased plasma concentrations of IGF-I and leptin compared with those of control cows. The revised quantitative insulin sensitivity check index was lower for feed-restricted cows (period 2) than for control cows. Compared with the NEB in period 1, the changes due to the deliberately induced NEB (period 2) in hormones were less pronounced. At the end of the 3-wk feed restriction, the mRNA abundance of IGF-I, IGFBP-1, -2, -3, and insulin receptor was increased as compared with the control group. The different effects of energy deficiency at the 2 stages in lactation show that the endocrine regulation changes qualitatively and quantitatively during the course of lactation.