Hypothyroidism is associated with increased myostatin expression in rats.

Besides its key role in the regulation of muscle growth during development, myostatin also appears to be involved in muscle homeostasis in adults, and its expression is upregulated during muscle atrophy. Since muscle physiology is greatly influenced by thyroid status, and the myostatin promoter region contains several putative thyroid hormone response elements, in the present study we examined the possible role of thyroid hormones in the regulation of myostatin gene expression. Adult male rats were made either hypothyroid or hyperthyroid by means of administration of 0.1% amino- triazole (AMT) in drinking water for 4 weeks, or daily injections of Levo-T4 (L-T4) (100 microg/rat) for 3 weeks, respectively. At the end of the treatment period, both myostatin mRNA and protein content were increased in AMT-treated rats in relation to control rats. In contrast, no changes in myostatin mRNA levels were detected in L-T4-treated rats. The role of thyroid hormones in the regulation of myostatin expression was also investigated in C2C12 cells in vitro. Treatment of C2C12 cells with thyroid hormones stimulated their differentiation into multinucleated myotubes, but did not induce any change in myostatin mRNA abundance. In all, our findings demonstrate that myostatin expression is increased in hypothyroid rats, thus supporting a possible role for this factor in the pathogenesis of the muscle loss that may occur in hypothyroidism.

Activation of growth hormone receptor delivers an antiapoptotic signal: evidence for a role of Akt in this pathway.

A signaling pathway was delineated by which GH promotes cell survival. Experiments were performed in human leukemic cells (HL-60) and Chinese hamster ovary (CHO) cells. In HL-60 cells, GH treatment reduced starvation-induced cell death. In contrast, when HL-60 cells were treated with an anti-GH antibody, cell survival was sharply reduced. In CHO cells stably expressing either the wild-type (wtGHR) or a truncated form (delta454GHR) of the GH receptor in which GH induces a sustained activation of the receptor-associated tyrosine kinase JAK2, we found that GH stimulation inhibited programmed cell death induced by withdrawal of survival factors. This effect was enhanced in cells expressing the truncated form. In contrast, GH did not affect cell survival in CHO cells transfected with either the empty vector or a mutated GHR unable to transduce the signal (4P/AGHR). We also showed that the inhibitory action of GH on apoptosis is probably mediated via stimulation of the serine-threonine kinase Akt, as 1) GH treatment induces a prompt phosphorylation of Akt; and 2) GH effects on cell survival are abolished by transfection of an Akt mutant that exhibits dominant negative function. Experiments with pharmacological inhibitors demonstrated that GH-induced Akt phosphorylation is dependent on phosphoinositide 3-kinase activation. In contrast, we found no changes in Bcl-2 levels secondary to GHR activation.

Deprivation of growth hormone-releasing hormone early in the rat's neonatal life permanently affects somatotropic function.

This work investigated in rats whether passive immunization against the endogenous GHRF in the early postnatal period led to permanent alterations of somatotropic function, similar to those observed in several human growth disorders, e.g. constitutional growth delay (CGD). On postnatal days 1, 2, 4, 6, 8, and 10, rats were given an anti-GHRF-serum (GHRH-Ab, 100 microliters/rat, sc) and were tested 1, 30, and 60 days after this treatment for basal and GHRH-stimulated GH secretion both in vivo and in vitro. GHRH-Ab reduced both basal and GHRF-stimulated GH secretion at all intervals and induced marked and chronic impairment of growth rate. The following differences were observed in the GHRH-Ab treated rats compared to normal rabbit serum-treated controls: 1) GH biosynthesis (incorporation of L-[3H]leucine into the electrophoretic band of GH): reduction of about 70%, 1 day but not 30 days after treatment; 2) Pituitary weight: significant reduction in absolute weight (30-40%) at all posttreatment intervals, and relative weight, 1 and 30 days after treatment. 3) Pituitary GH concentration: significant reduction in GH content (about 40%) but not concentration, at all posttreatment intervals; 4) Percentage of somatotrophs (immunocytochemistry): about 40% reduction 1 day, but not 30 and 60 days after treatment; 5) Hypothalamic somatostatin messenger RNA (mRNA) levels in situ hybridization): selective reduction (40%) in the periventricular nucleus 1 day but not 30 days after treatment; 6) Hypothalamic somatostatin cell number (immunocytochemistry): no significant changes in any hypothalamic area at any interval; 7) Pituitary somatostatin binding (in situ autoradiography): significant reduction, 1 day and 30 days after treatment; 8) Somatostatin inhibition of GH release "in vitro": somatostatin effect on GH release was reduced 30 days after treatment. These and previous data indicate that: 1) Transient deprivation of GHRF in the immediate postnatal period of the rat leads to permanent impairment of growth rate and somatotropic function; 2) GHRF deficiency itself or through reduction of GH secretion impairs somatostatin functions temporarily in the hypothalamus and permanently in the pituitary; 3) This rat model may mimic some forms of growth disorders in humans and holds promise as useful tools for investigating the underlying pathophysiological mechanisms.

Functional interrelationships between adrenergic and opioid systems in the neuroregulation of growth hormone secretion in infant rats.

Abstract Functional interrelationships between hypothalamic adrenergic and opioid systems were studied in 10-day-old male and female rats. Either clonidine (150 mug/kg, sc), an alpha(2)-adrenoceptor agonist, or FK 33-824 (1 mg/kg, sc), a synthetic analog of met-enkephalin, increased plasma growth hormone (GH) levels, the increment being significantly higher with FK 33-824 than with clonidine. Pharmacologic blockade of opioid receptors with naloxone (5 mg/kg, sc) did not modify either basal GH levels, or the GH response to clonidine, whereas blockade of alpha(2)-adrenoceptors with yohimbine (2.5 mg/kg, sc) reduced basal GH levels and partially counteracted the FK 33-824-induced GH rise. Clonidine (150mu/kg, sc, twice daily) administered from postnatal day 5 to 9, increased basal GH levels and pituitary GH content. In these pups, acute administration of clonidine failed to further release GH, but the GH response to acute administration of FK 33-824 was significantly enhanced. A 5-day treatment with FK 33-824 (1 mg/kg, sc, twice daily), neither modified basal GH levels, nor pituitary GH content. Under these conditions, the in vivo GH response to an FK 33-824 challenge was significantly enhanced, and the response to clonidine was preserved. Pituitaries from FK 33-824-pretreated rats were hyperresponsive to GH-releasing hormone (10(-7) M). In summary, our data indicate that in rat pups: 1) two separate components i.e. one adrenergic, the other extra-adrenergic, subserve the GH-releasing effect of opioid peptides; 2) in contrast to short-term stimulation of alpha(2)-adrenoceptors, stimulation of opioid receptors does not trigger GH synthesis or induce down-regulation or tolerance; 3) short-term opioid stimulation does not affect an alpha(2)-adrenergic challenge, but sensitizes to an opioid challenge.

Effect of enhancement of cholinergic tone on the growth hormone response to acute hyperglycaemia or thyrotropin-releasing hormone in dogs.

The effect of enhancement of cholinergic tone by pyridostigmine on the growth hormone (GH) response to thyrotropin-releasing hormone (TRH) or glucose-induced acute hyperglycaemia was tested in six adult unanaesthetized beagle dogs. Both TRH (5µg/ kg iv) and glucose (2 g/kg orally) did not significantly alter baseline GH levels but reduced the GH response to GH-releasing hormone (GHRH) (2 µg/kg iv), although this effect was more clear-cut with TRH than with glucose. Pretreatment with pyridostigmine (2 mg/kg orally) counteracted the inhibitory effect of hyperglycaemia on the GHRH-induced GH release, but had no effect on the inhibition induced by TRH. In summary, these results indicate that: 1) acute hyperglycaemia and TRH play an inhibitory role on GHRH-stimulated GH secretion in dogs; 2) the inhibitory effect of acute hyperglycaemia is mediated via hypothalamic cholinergic neurotransmission, whereas other neurotransmitter pathways would be. involved in the effect of TRH.

Expression of growth hormone receptor in the human brain.

This study was designed to investigate the presence of growth hormone receptor (GHR) expression in the human brain tissue, both normal and tumoral, as well as in the human glioblastoma cell line U87MG. Reverse transcription-polymerase chain reaction revealed the presence of GHR mRNA in all brain samples investigated and in U87MG cells. GHR immunoreactivity was also detected in this cell line using both immunocytochemistry and western blotting. All together, our data demonstrate the existence of GHR expression within the central nervous system (CNS), thus supporting a possible role for GH in the CNS physiology.

Proteolytic processing of human growth hormone (GH) by rat tissues in vitro: influence of sex and age.

Although a wealth of evidence exists indicating that proteolytic cleavage can enhance the biological activity of the growth hormone (GH) molecule, the mechanisms responsible for the generation of GH fragments are not completely understood. In the present work we investigated the ability of different rat tissues to cleave 22 kDa GH, as well as the influence of sex and age, the two major physiological regulators of GH secretion on this process. Our results show that tissue homogenates obtained from rat liver, skeletal muscle or adipose tissue (three well-documented target organs for the hormone) are able to cleave 22K-GH, while the hormone is resistant to cleavage by rat brain homogenates. This process is rather selective for 22K-GH, since the 20 kDa GH variant exhibits stability to degradation by all tissue homogenates investigated. Moreover, only a minor fraction of 22 kDa GH is cleaved under our experimental conditions, suggesting that GH microheterogeneity within the 22 kDa range may also determine hormone susceptibility. Finally, we also found that 22K-GH processing shows important age-related changes (the greatest intensity observed in 4-day-old pups), while no gender-related differences exist in any of the tissues investigated.

Myostatin regulates cell survival during C2C12 myogenesis.

During the myogenic process in vitro, proliferating myoblasts withdraw irreversible from the cell cycle, acquire an apoptosis-resistant phenotype, and fuse into mature myotubes. The key factor regulating both myocyte cell cycle exit and viability during this transition is the the cyclin-dependent kinase inhibitor p21(cip1). Here we show that the expression of myostatin, a TGF-beta superfamily member known to act as a negative regulator of muscle growth, is upregulated in the course of C2C12 cells myogenesis. We also show that transient transfection of C2C12 myobasts with an expression vector encoding mouse myostatin cDNA efficiently inhibits cell proliferation. Paradoxically, myostatin cDNA overexpression also enhances the survival of differentiating C2C12 myocytes, probably by a mechanism involving, at least in part, upregulation of p21(cip1) mRNA. Our results suggest that myostatin role in myogenesis is more complex than initially suggested and involves another level of regulation apart from inhibition of myoblast proliferation.

Studies of growth hormone secretion in calorically restricted dogs: effect of cholinergic agonists and antagonists, glucose and thyrotropin-releasing hormone.

The effect of caloric restriction (CR) on the growth hormone (GH) response to compounds reportedly capable of acting via somatostatinergic influences, i.e. cholinergic agonist or antagonist drug, glucose or thyrotropin-releasing hormone (TRH), administered alone or combined with GH-releasing hormone (GHRH), was evaluated in dogs. Eight beagle dogs, aged 4-5 years, underwent a 26-day period of increasing CR; they were evaluated either under basal conditions or starting from day 13 of CR, according to a schedule which allowed the mean length of CR to be similar among individual tests. CR resulted in a significant increase in basal GH levels, and starting from day 13 in a significant decrease in plasma somatomedin C levels; plasma glucose levels were significantly diminished on day 13 of CR and then remained unaltered. Administration of GHRH (GHRH1-44, 2 micrograms/kg, i.v.) induced a rise in plasma GH levels strikingly higher during CR than under basal conditions. Pyridostigmine (2 mg/kg orally), a muscarinic cholinergic agonist reportedly capable of restraining hypothalamic release of somatostatin (SS), enhanced the GH response to GHRH under basal conditions, but failed to do so during CR. Conversely, pirenzepine (0.6 mg/kg, i.v.), a muscarinic cholinergic antagonist, abolished the GHRH-induced GH rise under basal conditions, but only reduced it during CR. Only by doubling the dose of pirenzepine was complete inhibition of the GHRH-induced GH rise effected. Glucose alone (2 g/kg, p.o.) failed to modify basal GH secretion either before or during CR, but significantly inhibited the GHRH-induced GH rise either before or during CR.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined administration of growth-hormone-releasing hormone and clonidine restores defective growth hormone secretion in old dogs.

We have studied in old dogs the effects of short-term administration of growth hormone (GH)-releasing hormone (GHRH) alone or co-administered with clonidine (CLO), an alpha 2-adrenergic agonist, on the GH secretory pattern (cluster analysis), and GH responsiveness to an acute GHRH or GHRH + CLO challenge and plasma somatomedin C (SMC) levels. Dogs were given either GHRH alone twice daily for 10 days (treatment 1) or combined GHRH + CLO both given twice daily (treatment 2) or GHRH + CLO given once daily (treatment 3). Animals were sampled from 09.00 to 15.00 h, at 10-min intervals, both before and 14 h after treatments. At the end of the 6-hour sampling period, dogs were challenged with simultaneous administration of GHRH and CLO, while they were tested with GHRH alone on the morning of the following day. In dogs undergoing treatment 1, acute administration of GHRH or GHRH + CLO elicited mean GH peak responses higher than before treatment, but none of the GH secretory indices were modified during the 6-hour sampling period, except for the increase in mean GH peak amplitude. In dogs undergoing treatment 2, acute administration of GHRH elicited a mean GH peak response higher than that before treatment, whereas administration of GHRH + CLO induced a mean GH peak response not different from that elicited by GHRH + CLO before treatment or by GHRH alone after treatment. However, this treatment significantly augmented the frequency of spontaneous bursts of GH secretion, the mean GH peak amplitude and the total peak area.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of Pit-1 gene expression and Pit-1 content with proliferation and differentiation in human myeloid leukemic cells.

The transcription factor pituitary-1 (Pit-1) is a homeodomain-containing protein that is expressed mainly in the pituitary, where it drives the expression of growth hormone, prolactin, and thyroid-stimulating hormone beta chain genes. In addition, Pit-1 is required for adequate pituitary cell growth and may be involved in the pathogenesis of pituitary adenomas. Pit-1 expression has been also reported in nonpituitary tissues, where it might be involved in the control of cell proliferation. In order to elucidate such a possibility, we have investigated the changes in both Pit-1 mRNA and Pit-1 immunoreactivity in HL-60 cells following the addition of several differentiating agents. Our results show that while high Pit-1 levels are found in exponentially growing HL-60 cells, a significant decrease occurs after induction of cells to differentiate along the macrophage lineage with 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast no changes were observed when cells were treated with interferon-alpha, which also induces differentiation of HL-60 cells that, at odds with TPA, is not accompanied with growth arrest. In all, these findings suggest that Pit-1 expression is specifically associated with proliferation in HL-60 cells, thus supporting the idea that one of the functions of nonpituitary Pit-1 may be the control of cell proliferation.

Role of growth hormone receptor in HL-60 cell survival.

Although it is presently well established that locally produced growth hormone (GH) plays a major role in the regulation of survival mechanisms in hemopoietic cells, the responsible mechanisms are poorly understood, and the involvement of the GH receptor (GHR) has not even been demonstrated to date. In this work we investigated the presence of GHR in the human promyelocytic leukemia cell line HL-60, as well as the ability of GH treatment to stimulate both GHR and survival signaling pathways downstream GHR. Our results demonstrate that (1) both GHR mRNA and GHR immunoreactivity are present in HL-60 cells; (2) GH treatment results in an increase in the phosphorylation of the GHR-associated Jak2 and Stat3 proteins, indicating the ability of the hormone to induce receptor activation; and (3) activation of GHR increases the activity of Akt, a serine/threonine kinase that plays a prominent role in the regulation of cell survival. Taken together, these results demonstrate that GHR activation promotes survival of HL-60 cells, thus suggesting that GH plays a major role in the regulation of cell survival in the hemopoietic system, via an autocrine/paracrine mechanism.

La miostatina: un regulador autocrino/paracrino del desarrollo muscular / Myostatin: an autocrine/paracrine regulator of muscular development

La miostatina es una proteína perteneciente a la familia del factor de crecimiento de transformación (TGF)-ß, que desempeña un papel fundamental en el control del desarrollo muscular. Como ocurre con el resto de miembros de la familia del TGF-ß, la miostatina se sintetiza en forma de un precursor inactivo que ha de tener un procesamiento proteolítico para dar lugar a la forma madura. La miostatina se expresa de forma casi exclusiva en el músculo esquelético donde actúa de forma autocrina/paracrina al inhibir el desarrollo muscular. En ratones, el bloqueo de la miostatina produce un marcado aumento de la masa muscular y una disminución de la adiposidad. Este efecto sobre el tejido adiposo es tan marcado que el bloqueo de la miostatina es incluso capaz de revertir la obesidad en diversas cepas de ratones. Debido a estas acciones, se está comenzando a estudiar el uso de fármacos capaces de bloquear la miostatina para la prevención y el tratamiento de la obesidad, la diabetes tipo 2, y en enfermedades en las que es necesario favorecer el anabolismo muscular (como ocurre en algunas distrofias musculares o en los cuadros de caquexia) (AU)

Myostatin is a protein belonging to the transforming growth factor (TGF)-ß family, which plays a major role in controlling muscular development. As occurs with other members of the TGF-ß family, myostatin is synthesised as an inactive precursor that needs to undergo proteolytic processing to give rise to the mature peptide. Myostatin is almost exclusively expressed in skeletal muscle, where it acts in an autocrine/paracrine fashion to inhibit muscle growth. In mice, myostatin blockade results in a dramatic increase in muscle mass and decreased adiposity. The effect on adipose tissue is so marked that myostatin blockade is even capable of reverting obesity in several strains of obese mice. Because of these actions, the use of myostatin-blocking agents has been proposed as a new strategy in the prevention or treatment of obesity and type 2 diabetes, as well as in diseases in which muscular anabolism needs to be stimulated (such as some muscular dystrophies and wasting conditions) (AU)