Molecular identification and developmental expression patterns of growth hormone and its receptors in yellowtail kingfish (Seriola lalandi).

In fish and other vertebrates, growth hormone (GH) is an essential polypeptide required for normal growth and development. In an attempt to understand growth regulation in yellowtail kingfish (YTK), the full-length cDNA sequences encoding gh and its receptors (ghr1 and ghr2) were cloned, characterized and the expression profiles of these three genes were investigated during embryonic development. The full-length cDNA sequences of GH and its receptors were obtained by RT-PCR combined with RACE methord. YTK gh cDNA sequence was 852 base pairs (bp) that comprised an open reading frame (ORF) of 615 bp encoding a 204-amino acids (aa) precursor. The preprohormone compassed a signal peptide (17 aa) and the mature peptide (187 aa). YTK GHR1 protein consisted of a signal peptide (28 aa), an extracellular domain (222 aa), a single transmembrane domain (23 aa) and an intracellular domain (361 aa). GHR2 protein included 18 aa, 223 aa, 23 aa, and 321 aa, respectively. Tissue distribution analysis showed that the maximal level of gh expression was observed in the pituitary, and ghr1 mRNA was mainly detected in the liver, while ghr2 transcripts were most abundant in the gonad. Moreover, both ghr1 and ghr2 mRNAs were expressed in all embryonic stages and displayed different gene expression profiles. Overall, these results provide initial evidences for the involvement of the GH/GHR system in the early ontogeny of yellowtail kingfish.

Expression and localisation of FSHR, GHR and LHR in different tissues and reproductive organs of female yaks.

BACKGROUND: This study aimed to investigate the expression and localisation of fol-licle stimulating hormone receptor/growth hormone receptor/luteinising hormone receptor (FSHR/GHR/LHR) in different tissues and examine the regulatory effects of FSHR/GHR/LHR in the reproductive organs of female yaks during luteal phase. MATERIALS AND METHODS: The quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry assays were utilised to analyse the expression and localisation of FSHR/GHR/LHR in different tissues on female yaks. RESULTS: The qRT-PCR results showed that the mRNA expressions of FSHR/GHR/ /LHR were significantly different in the non-reproductive organs (p < 0.01); the highest expression level was observed in the kidney, cerebellum and lung, whereas the lower expression level was observed in the liver and spleen. Im-munohistochemistry assay results showed that FSHR/GHR/LHR were located in kidney tubules, Purkinje cells, cerebellar medulla, alveolar cells and hepato-cytes. In addition, the expression levels of FSHR and GHR were considerably higher than LHR in the reproductive organs of female yaks during luteal phase (p < 0.01). FSHR/GHR/LHR were located in cardiac muscle cells, cerebellar medulla, and theca cell lining of reproductive organs. Furthermore, the expression level of FSHR was higher than those of GHR and LHR in all examined tissues. CONCLUSIONS: Therefore, the expression and localisation of FSHR/GHR/LHR possibly helped to evaluate the effects of them in tissue specific expression on female yaks, investigate the function and mechanism of FSHR/GHR/LHR in the reproductive organs of female yaks during luteal phase. (Folia Morphol 2018; 77, 2: 301-309).

Reproductive parameters of double transgenic zebrafish (Danio rerio) males overexpressing both the growth hormone (GH) and its receptor (GHR).

Growth hormone (GH) transgenesis presents a high potential application in aquaculture. However, excess GH may have serious consequences due to pleiotropic actions. In order to study these effects in zebrafish (Danio rerio), two transgenic lines were developed. The first expresses GH ubiquitously and constitutively (F0104 line), while the second expresses the GH receptor in a muscle-specific manner (Myo-GHR line). Results from the F0104 line showed accelerated growth but increased reproductive difficulties, while Myo-GHR did not show the expected increase in muscle mass. Since the two lines appeared to display complementary characteristics, a double transgenic (GH/GHR) was created via crossing between them. This double transgenic displayed accelerated growth, however reproductive parameters remained uncertain. The objective of the present study was to determine the reproductive capacity of males of this new line, by evaluating sperm parameters, expression of spermatogenesis-related genes, and reproductive tests. Double transgenics showed a strong recovery in almost all sperm parameters analyzed when compared to the F0104 line. Gene expression analyses revealed that Anti-Müllerian Hormone gene (amh) appeared to be primarily responsible for this recovery. Reproductive tests showed that double transgenic males did not differ from non-transgenics. It is possible that GHR excess in the muscle tissues of double transgenics may have contributed to lower circulating GH levels and thus reduced the negative effects of this hormone with respect to reproduction. Therefore, it is clear that GH-transgenesis technology should take into account the need to obtain adequate levels of circulating hormone in order to achieve maximum growth with minimal negative side effects.

[Preparation of Transmembrane Fragments Growth Hormone Receptor GHR in a Cell-Free Expression System for Structural Studies].

Growth hormone somatotropin and its membrane receptor GHR, belonging to a superfamily of the type I receptors possessing tyrosine kinase activity, are involved in the intercellular signal transduction cascade and regulate a number of important physiological and pathological processes in humans. Binding with somatotropin triggers a transition of GHR between two alternative dimer states, resulting in an allosteric activation of JAK2 tyrosine kinase in the cell cytoplasm. Transmembrane domain of GHR directly involved in this complex conformational transition. It has presumably two dimerization interfaces corresponding to the "unliganded" and the active state of GHR. In order to study the molecular basis of biochemical signal transduction mechanism across the cell membrane, we have developed an efficient cell-free production system of a TM fragment of GHR, which contains its TM domain flanked by functionally important juxtamembrane regions (GHRtm residues 254-298). The developed system allows to obtain -1 mg per 1 ml of reaction mixture of 13C- and 15N-isotope-labeled protein for structural and dynamic studies of the GHR TM domain dimerization in the membrane-mimicking medium by high-resolution heteronuclear NMR spectroscopy.

Dietary methionine effects on IGF-I and GHR mRNA expression in broilers.

This study aimed to evaluate liver and breast muscle insulin-like growth factor I (IGF-I) and growth hormone receptor (GHR) gene expression between broilers fed different methionine levels and sources. Broiler chicks were 22 to 42 days old, distributed in 5 treatments (control diet, DL1 - 0.08% DL-methionine, DL2 - 0.24% DL-methionine, MHA-FA1 - 0.11% methionine hydroxy analogue-free acid, and MHA-FA2 - 0.33% methionine hydroxy analogue-free acid). The broilers were euthanized by cervical dislocation. RNA was extracted from liver and breast muscle, followed by cDNA synthesis and amplification using qRT-PCR. DL2 methionine supplementation provided best animal performance results. GHR and IGF-I gene expression in the muscle tissue was not affected by methionine supplementation. IGF-I gene expression in the liver was higher in animals fed methionine supplementation than in animals fed control diet. IGF-I mRNA levels in broilers fed DL2 were greater than DL1 (1.56 vs 0.97 AU) and greater than MAH-FA1 and MAH-FA2. Broilers fed DL2 increased significantly GHR gene expression in the liver than animals fed the control diet. Addition of methionine improved animal performance by stimulating synthesis and release of growth factor.

Growth hormone treatment induces mammary gland hyperplasia in aging primates.

The decline of growth hormone (GH) and insulin-like growth factor I (IGF-I) production during aging has been likened to the decrease in gonadal steroids in menopause. The repletion of GH/IGF-I levels in aging individuals is suggested to restore the lean tissue anabolism characteristic of youth. In addition to anabolic effects on musculo-skeletal tissues, GH also stimulates mammary glandular growth in some species, although its effects on primate mammary growth remain unclear. Some clinical observations implicate GH in human mammary growth, for example, gynecomastia occurs in some children treated with GH (ref. 6), and tall stature and acromegaly are associated with an increased incidence of breast cancer. To investigate the effects of GH/IGF-I augmentation on mammary tissue in a model relevant to aging humans, we treated aged female rhesus monkeys with GH, IGF-I, GH + IGF-I or saline diluent for 7 weeks. IGF-I treatment was associated with a twofold increase, GH with a three- to fourfold increase, and GH + IGF-I with a four'-to fivefold increase in mammary glandular size and epithelial proliferation index. These mitogenic effects were directly correlated with circulating GH and IGF-I levels, suggesting that either GH or its downstream effector IGF-I stimulates primate mammary epithelial proliferation.

Expression, cellular distribution, and heterogeneity of growth hormone in the chicken cerebellum during development.

Although growth hormone (GH) is mainly synthesized and secreted by pituitary somatotrophs, it is now well established that the GH gene can be expressed in many extrapituitary tissues, including the central nervous system (CNS). Here we studied the expression of GH in the chicken cerebellum. Cerebellar GH expression was analyzed by in situ hybridization and cDNA sequencing, as well as by immunohistochemistry and confocal microscopy. GH heterogeneity was studied by Western blotting. We demonstrated that the GH gene was expressed in the chicken cerebellum and that its nucleotide sequence is closely homologous to pituitary GH cDNA. Within the cerebellum, GH mRNA is mainly expressed in Purkinje cells and in cells of the granular layer. GH-immunoreactivity (IR) is also widespread in the cerebellum and is similarly most abundant in the Purkinje and granular cells as identified by specific neuronal markers and histochemical techniques. The GH concentration in the cerebellum is age-related and higher in adult birds than in embryos and juveniles. Cerebellar GH-IR, as determined by Western blot under reducing conditions, is associated with several size variants (of 15, 23, 26, 29, 35, 45, 50, 55, 80 kDa), of which the 15 kDa isoform predominates (>30% among all developmental stages). GH receptor (GHR) mRNA and protein are also present in the cerebellum and are similarly mainly present in Purkinje and granular cells. Together, these data suggest that GH and GHR are locally expressed within the cerebellum and that this hormone may act as a local autocrine/paracrine factor during development of this neural tissue.

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia.

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL177 and PRL188; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na+/Cl- cotransporter and a Na+/H+ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na+/K+/2Cl- cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.

Expression profiles of somatotropic axis genes in lines of chickens divergently selected for 56-day body weight.

The objective of this study was to evaluate mRNA expression of somatotropic axis genes in chickens divergently selected for high (HWS) or low (LWS) body weight at 56 days of age. Gene expression was measured on days 16, 18, and 20 of incubation, day of hatch, and days 3, 7, 28, and 56 posthatch. Pituitary growth hormone mRNA raised from prehatch to posthatch, with a similar profile in both lines. Liver growth hormone receptor (GHR) mRNA was high during embryogenesis, declined to low levels at day 3 posthatch, and then increased to day 56. Expression of liver insulin-like growth factor 1 (IGF-1) mRNA increased sharply by day 28 in line HWS and day 56 in line LWS. Pectoralis major muscle GHR mRNA was greater in line LWS than HWS. Muscle IGF-1 mRNA declined during embryogenesis, increased posthatch, and declined after day 7. IGF-1 mRNA was 1,000-fold greater in embryonic muscle than embryonic liver. Muscle IGF-1 receptor mRNA was greater in line LWS than HWS posthatch. These results demonstrate that genetic selection for high or low body weight has altered the expression profiles of somatotropic axis genes in a line-, age-, and tissue-specific manner.

The Role of Growth Hormone Receptor Isoforms and Their Effects in Bone Metabolism and Skeletal Fragility.

Acromegaly and Growth Hormone Deficiency (GHD) are associated with skeletal fragility and with an increased prevalence of Vertebral Fractures (VFs). In the most recent years, several authors tried to investigate surrogate markers that may predict the risk of bone fragility in these endocrine disorders. The aim of this review is to evaluate the role of GH receptor polymorphisms in skeletal fragility in patients affected by GHD and acromegaly. In fact, until now, two different isoforms of the GH Receptor (GHR) were described, that differ for the presence or the absence of transcription of the exon 3 of the GHR gene. Both the isoforms produce a functioning receptor, but the exon 3-deleted isoforms (d3-GHR) has a higher sensitivity to endogenous and recombinant GH as compared to the full-length isoform (fl-GHR).

Heterogeneity of Growth Hormone Receptor Expression in Craniopharyngioma-Implications for Surgical Strategy.

BACKGROUND: Recent molecular investigations for craniopharyngiomas have investigated possible predictive biologic markers. Growth hormone receptor (GHR) is thought to be involved in tumor aggressiveness, and high expression of GHR is associated with shorter duration of postoperative stable disease. CASE DESCRIPTION: A 27-year-old man with a large suprasellar tumor underwent an inexplicable clinical course. Transsphenoidal surgery achieved gross total removal of the tumor. Histologic diagnosis was adamantinomatous craniopharyngioma, and immunohistochemistry revealed very low GHR expression. He was discharged with multiple hormonal supplements except for growth hormone. Recurrence was detected 18 months later, and removal of the second tumor was performed with coagulation of the superior surface of the remaining pituitary gland. Growth hormone supplementation was started in the postoperative period, and he returned to work. However, a further recurrence was detected. Head magnetic resonance imaging showed almost the same pattern of tumor recurrence as preoperative imaging of the second surgery, and simultaneous removal of the tumor and the normal pituitary gland was performed. Reevaluation of histology revealed no morphologic differences between the first and the third surgical specimens, but immunohistochemical staining for GHR showed diffuse high expression in the third specimen. The difference was thought to reflect the heterogeneity of GHR, and appearance of histologic hot spots greatly affected the postoperative prognosis. CONCLUSIONS: Extensive removal of the possible tumor bed may be necessary for patients requiring growth hormone supplementation even after gross total removal of craniopharyngioma.

Divergent personalities influence the myogenic regulatory genes myostatin, myogenin and ghr2 transcript responses to Vibrio anguillarum vaccination in fish fingerlings (Sparus aurata).

Myogenic regulators of muscle development, metabolism and growth differ between fish species in a context-specific manner. Commonly, the analysis of environmental influences on the expression of muscle-related gene regulators in teleosts is based on differences in swimming performance, feeding behaviour and stress-resistance, but the evaluation of behavioural phenotyping of immune and stress-related responsiveness in skeletal muscle is still scarce. Here we challenge proactive and reactive fingerlings of gilthead sea bream (Sparus aurata), one of the most commonly cultured species in the Mediterranean area, with highly pathogenic O1, O2α and O2ß serotypes of Vibrio anguillarum, a widespread opportunistic pathogen of marine animals, to analyse skeletal muscle responses to bath vaccination. Transcripts related to inflammation (interleukin 1ß, il1ß; tumour necrosis factor-α, tnfα; and immunoglobulin M, igm), and muscle metabolism and growth (lipoprotein, lpl; myostatin, mstn-1; myogenin; and growth hormone receptors type I and II, ghr1 and ghr2, respectively) were analysed. Biochemical indicators of muscle metabolism and function (creatine kinase, CK, aspartate aminotransferase, AST; esterase activity, EA; total antioxidant status, TAC and glucose) were also determined. Our results indicate that proactive, but not reactive, fish respond to Vibrio vaccination by increasing the expression levels of mstn-1, myogenin and ghr2 transcripts at short-/medium- term (1 to 3 days' post vaccination). No effect of vaccination was observed in immune indicators or biochemical parameters in either phenotypes, except for elevated levels of EA in reactive fish one-week post vaccination. This suggests that behavioural divergence should be taken into account to evaluate the crosstalk between immune, metabolic and growth processes in muscle of immune-challenged fish.

Uterine and hepatic gene expression in relation to days postpartum, estrus, and pregnancy in postpartum dairy cows.

The somatotropic axis consisting of growth hormone, the growth hormone receptor (GHR) insulin-like growth factor (IGF)-I, and IGF binding proteins changes with the stage of lactation and nutrition of the cow and may be 1 mechanism through which lactation and nutrition affect the establishment of pregnancy. The objective of this study was to quantify GHR, IGF-I, and IGF binding protein-2 (IGFBP-2) mRNA in liver and uterine endometrial tissue at 4 stages of lactation (40, 80, 120, and 160 days in milk) and around the time of artificial insemination. Estrus was synchronized with GnRH and PGF2alpha, and cows were inseminated 12 h after estrus. Uterine biopsies were collected immediately before the second injection of PGF2alpha (before estrus), at the initiation of standing estrus, and 4 d after estrus. Liver biopsies were collected once on 4 d after estrus. The abundance of GHR, IGF-I, and IGFBP-2 mRNA in liver and uterus was determined by real-time quantitative PCR. The amount of liver IGF-I mRNA was positively correlated with plasma IGF-I concentrations. Cows that became pregnant after AI had more GHR and IGFBP-2 mRNA in their liver than cows that did not become pregnant. There was no effect of DIM or pregnancy status on abundance of uterine mRNA; however, uterine GHR and IGF-I mRNA was most abundant at estrus. In summary, cows at different stages of lactation or with different pregnancy statuses had similar quantities of uterine mRNA. In contrast, liver quantities of mRNA differed relative to pregnancy status. These data provide evidence that liver indices of metabolic state may be indicative of pregnancy success.

Current Strategies of Endocrine Therapy in Elderly Patients with Breast Cancer.

Currently, the growing population of the elderly is one of biggest problems in terms of increase in geriatric diseases. Lack of data from large prospective studies on geriatric breast cancer patients often makes it difficult for clinicians to make treatments decisions for them. Because both benefit and risk of treatment should be taken into account, treatment is usually determined considering life expectancy or comorbidities in elderly patients. Treatment of breast cancer is differentiated according to histologic classifications, and hormone therapy is even adopted for patients with metastatic breast cancer if tumor tissue expresses hormone receptors. Endocrine therapy can offer great benefit to elderly patients considering its equivalent efficacy to chemotherapy with fewer toxicities if it is appropriately used. Aromatase inhibitors are usually prescribed agents in hormone therapy for elderly breast cancer patients due to their physiology after menopause. Here, endocrine therapy for elderly patients with breast cancer in neoadjuvant, adjuvant, and palliative setting is reviewed along with predictive adverse events resulting from the use of hormone agents.

Growth hormone in vascular pathology: neovascularization and expression of receptors is associated with cellular proliferation.

Vascular tumours are common lesions of the skin and subcutaneous tissue, but also occur in many other tissues and internal organs. The well-differentiated tumours consist of irregular anastomosing, blood-filled vascular channels that are lined by variably atypical endothelial cells. The less differentiated tumours may show solid strands and sheets, resembling carcinoma or lymphoma. Several growth factors, including basic fibroblast growth factor, transforming growth factors and vascular endothelial growth factor, play a role in tumour angiogenesis. Growth hormone (GH) is mitogenic for a variety of vascular tissue cells, including smooth muscle cells, fibroblasts and endothelial cells and exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific membrane-bound receptors, which trigger a phosphorylation cascade resulting in the modulation of numerous signalling pathways and of gene expression. Essential to the initiation of a cellular response to GH, the presence of receptors for this hormone may predict the adaptation of tumour cells resulting from GH exposure. To address the site/mode of action through which GH exerts its effects, a well characterized monoclonal antibody, obtained by hybridoma technology from Balb/c mice immunized with purified rabbit and rat liver GH-receptor (GHR) and directed against the hormone binding site of the receptor, was applied, using the ABC technique to determine GHR expression in a panel of vascular tumours. The GHR was cloned from a rabbit liver cDNA library with the aid of an oligonucleotide probe based on a 19 residue tryptic peptide sequence derived from 5900 fold purified rabbit liver receptor. A total of 64 benign and malignant vascular tumours were obtained from different human organ sites, including the chest wall, skin, axillary contents, duodenum, female breast, abdomen, stomach, colon, lymph node, bladder, body flank and neck regions. The tumours were of the following pathological entities: Haemangioma (n = 12); haemangioendothelioma (n = 10); Castleman's disease (n = 3), haemangiopericytoma (n = 4); angiosarcoma, (n = 11), Kaposi's sarcoma with focal infiltration by lymphoma, HIV +ve (n = 7), Kaposi's sarcoma (n = 17). The endothelial cell marker CD-31 was used to establish endothelial cell characteristics and microvascular density. To delineate tumour cell growth, immunohistochemical analysis of cycling nuclear protein and of proliferating cell nuclear antigen, using Ki-67 and PCNA polyclonal antibodies respectively, was used to demonstrate proliferative indexes. Results show that, compared to their normal tissue counterparts, nuclear and cytoplasmic expression of GHR consistently result in strong receptor immunoreactivity in the highly malignant angiosarcomas and Kaposi's sarcomas and was localized in the cell membranes and cytoplasm, but strong nuclear immunoreactivity was also identified. The presence of intracellular GHR is the result of endoplasmic reticulum and Golgi localization. Nuclear localization is due to identical nuclear GHR-binding protein. Furthermore, there was a positive correlation of GHR immunoreactivity with neoplastic cellular proliferation and cycling, as measured by Ki-67 and PCNA. In conclusion, this study shows that GHR expression in vascular tumours is a function of malignancy and cancer progression. Malignant cells, which are highly expressive of the receptor, have a greater proliferation rate and thereby also higher survival rate compared to tumours expressing lower or minimal receptor level. The presence of GHR in endothelial cells of vascular neoplasm indicates that they are target cells and GH is of importance in the proliferation of vascular tumour angiogenesis. GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion and maintenance. The results support the hypothesis that GH is involved in the paracrine-autocrine mechanism, acting locally in regulating vascular tumour growth and will be useful for site-specific studies of the evolution of vascular cancers. The use of anti-GHR antibodies to block tumour progression is an intriguing possibility.

Somatostatin Agonist Pasireotide Inhibits Exercise-Stimulated Growth in the Male Siberian Hamster (Phodopus sungorus).

The Siberian hamster (Phodopus sungorus) is a seasonal mammal, exhibiting a suite of physiologically and behaviourally distinct traits dependent on the time of year and governed by changes in perceived day length (photoperiod). These attributes include significant weight loss, reduced food intake, gonadal atrophy and pelage change with short-day photoperiod as in winter. The central mechanisms driving seasonal phenotype change during winter are mediated by a reduced availability of hypothalamic triiodothyronine (T3), although the downstream mechanisms responsible for physiological and behavioural changes are yet to be fully clarified. With access to a running wheel (RW) in short photoperiod, Siberian hamsters that have undergone photoperiod-mediated weight loss over-ride photoperiod-drive for reduced body weight and regain weight similar to a hamster held in long days. These changes occur despite retaining the majority of hypothalamic gene expression profiles appropriate for short-day hamsters. Utilising the somatostatin agonist pasireotide, we recently provided evidence for an involvement of the growth hormone (GH) axis in the seasonal regulation of bodyweight. In the present study, we employed pasireotide to test for the possible involvement of the GH axis in RW-induced body weight regulation. Pasireotide successfully inhibited exercise-stimulated growth in short-day hamsters and this was accompanied by altered hypothalamic gene expression of key GH axis components. Our data provide support for an involvement of the GH axis in the RW response in Siberian hamsters.

Changes in skeletal muscle gene expression following clenbuterol administration.

BACKGROUND: Beta-adrenergic receptor agonists (BA) induce skeletal muscle hypertrophy, yet specific mechanisms that lead to this effect are not well understood. The objective of this research was to identify novel genes and physiological pathways that potentially facilitate BA induced skeletal muscle growth. The Affymetrix platform was utilized to identify gene expression changes in mouse skeletal muscle 24 hours and 10 days after administration of the BA clenbuterol. RESULTS: Administration of clenbuterol stimulated anabolic activity, as indicated by decreased blood urea nitrogen (BUN; P < 0.01) and increased body weight gain (P < 0.05) 24 hours or 10 days, respectively, after initiation of clenbuterol treatment. A total of 22,605 probesets were evaluated with 52 probesets defined as differentially expressed based on a false discovery rate of 10%. Differential mRNA abundance of four of these genes was validated in an independent experiment by quantitative PCR. Functional characterization of differentially expressed genes revealed several categories that participate in biological processes important to skeletal muscle growth, including regulators of transcription and translation, mediators of cell-signalling pathways, and genes involved in polyamine metabolism. CONCLUSION: Global evaluation of gene expression after administration of clenbuterol identified changes in gene expression and overrepresented functional categories of genes that may regulate BA-induced muscle hypertrophy. Changes in mRNA abundance of multiple genes associated with myogenic differentiation may indicate an important effect of BA on proliferation, differentiation, and/or recruitment of satellite cells into muscle fibers to promote muscle hypertrophy. Increased mRNA abundance of genes involved in the initiation of translation suggests that increased levels of protein synthesis often associated with BA administration may result from a general up-regulation of translational initiators. Additionally, numerous other genes and physiological pathways were identified that will be important targets for further investigations of the hypertrophic effect of BA on skeletal muscle.

Fetal- and tumor-specific regulation of growth hormone receptor messenger RNA expression in human liver.

Eight different 5'-untranslated region variants of the human growth hormone receptor (hGHR) mRNA have been identified in adult liver (V1-V8). We have compared the expression of two of these variants (V1 and V3) in several human fetal and postnatal tissues (including liver) as well as in hepatoblastomas (HBs) and hepatocellular carcinomas (HCCs). Using reverse transcription-PCR assays, followed by Southern blotting to confirm the specificity of the amplified fragments, we found that V3 was expressed in all fetal and postnatal liver (n = 13 fetal and 5 postnatal), kidney (n = 4 fetal and 4 postnatal), lung (n = 4 fetal and 2 postnatal), intestine (n = 8 fetal and 4 postnatal), skeletal muscle (n = 1 fetal and 1 postnatal), and adrenal (n = 1 fetal and 1 postnatal) samples. In contrast, V1 was expressed only in postnatal liver. We then screened for V1 and V3 in HBs (n = 17, ages 6-36 months, including 5 with paired normal liver), and HCCs (n = 4, ages 50-75 years, with paired normal liver). V1 was undetectable in 15 of 17 HBs, including all HBs paired with (V1-expressing) normal liver; the absence of V1 did not correlate with patient age, sex, HB subtype, +/- chemotherapy, exon 3-retaining and -deficient hGHR mRNA isoform pattern, or loss of heterozygosity at 11p, 1p, and 1q. The four HCCs showed marked (>20-fold; n = 2) or complete (n = 2) suppression of V1 as compared to paired normal liver. V3 was expressed in all HBs, HCCs, and paired normal livers. Interestingly, V3, but not V1, was detected in two Wilms' tumor and paired normal kidney specimens. Our findings suggest that, in the human, there is tissue-, fetal- and tumor-specific regulation of V1 hGHR mRNA.

Effect of growth hormone on the translocation of GLUT4 and its relation to insulin-like and anti-insulin action.

To elucidate the effect of growth hormone (GH) on the insulin signal transduction pathway leading to the translocation of glucose transporter-4 (GLUT4), we constructed Chinese hamster ovary cells that overexpressed GH receptor and GLUT4. Treatment with GH triggered GLUT4 translocation, and this translocation was completely inhibited by wortmannin. GH-induced GLUT4 translocation reached a maximum level after 30 min, and then gradually decreased and returned to the basal level after 2 h. Tyrosine phosphorylation of JAK2 also became maximal after 30 min and then gradually decreased. In contrast, GLUT4 translocation remained unchanged for 2 h after insulin treatment, and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) also remained constant for up to 2 h. Chronic GH treatment had almost no effect on insulin-stimulated Akt kinase activation and GLUT4 translocation. These results suggest that GH and insulin translocate GLUT4 in a similar manner, at least in part, and the difference in translocation depends on the difference in the tyrosine phosphorylation of JAK2 and IRS-1. The anti-insulin action of GH after chronic GH treatment does not appear to be mainly due to the inhibition of GLUT4 translocation.

Opposite regulation of IGF-I and IGF-I receptor mRNA and concomitant changes of GH receptor and IGF-II/M6P receptor mRNA in human IM-9 lymphoblasts.

Human IM-9 lymphoblasts synthesize IGF-I and express IGF-I receptors, IGF-II/M6P receptors and GH receptors. We have studied the regulation of mRNA expression of IGF-I, IGF-I receptors, IGF-II/M6P receptors and GH receptors in IM-9 cells upon serum-withdrawal and re-addition of serum. IM-9 cells were cultured in RPMI-1640 medium with or without serum for various periods of time. RNA was prepared using guanidinium thiocyanate and CsCl. Antisense riboprobes for human IGF-I, IGF-I receptor, IGF-II/M6P receptor, GH receptor and for comparison for human beta-actin were synthesized and labeled with 32P. Protected fragments of 379 bases and of 420 and 350 bases with the IGF-I receptor and with the IGF-I probe respectively and protected fragments of 670 bases and of 51 and 121 bases with the GH receptor and with the beta-actin probe were detected. For the human IGF-II/M6P receptor probe protected fragments of 260 bases were visualized in RNA samples. The amount of mRNA present in each lane (10 microgram total RNA) was determined by computed densitometry. The amount of IGF-mRNA expressed by IM-9 cells decreased rapidly (within two hours) and dramatically (more than 120%) after the withdrawal of serum and increased significantly (220%) after the re-addition of serum. This increase of IGF-I mRNA preceded the increase in cell number that was seen after 48 h of medium change. Conversely, the expression of IGF-I receptor mRNA and beta-actin mRNA increased by more than 250% after the withdrawal of serum within 2 and 8 h respectively, while GH receptor mRNA fell within 2-4 h. The expression of IGF-II/M6P receptor mRNA continued to increase throughout the duration of the cell culture experiment. We conclude that IGF-I and IGF-I receptor mRNAs are regulated in an opposite direction in serum-deprived IM-9 lymphoblasts. In addition, GH receptor mRNA expression parallels IGF-I mRNA expression.