Acciones neurotróficas de la hormona de crecimiento durante el desarrollo y envejecimiento cerebral / Neurotrophic actions of growth hormone during development and brain aging

En estudios previos hemos demostrado que la hormona de crecimiento(GH) induce la proliferación y diferenciación de células cerebrocorticalesembrionarias. El papel de la GH en el desarrollo de los oligodendrocitos(OD) y mielinización es poco conocido. Es este estudio se pretendió investigarsi la GH promueve el desarrollo de oligodendrocitos y los mecanismosde señalización implicados en este proceso. También se estudió, la acción deGH en remielinización cerebral durante el envejecimiento. Células cerebrocorticalesembrionarias se cultivaron en DMEM, 15% de FCS, durante6 horas, se mantuvieron 4 días en medio definido con FGFb (25ng/ml) y seexpusieron a GH (50ng/ml) durante 8 días. Los inhibidores de quinasas seañadieron 45 minutos antes que la GH. Ratas adultas (3 meses) y viejas (27meses) se trataron con GH subcutánea (150 µg/12hr) durante 7 días. Paraverificar el efecto de GH en desarrollo de oligodendrocitos y mielinogenesis,pre-OD y oligodendrocitos maduros se detectaron por inmunocitoquímicacon anticuerpos frente a O4 y proteína básica de mielina (MBP) respectivamente.MBP se analizó en cerebro por inmunohistoquímica. GHincrementó el número de células positivas a O4 (Control: 100%; GH:166±5%) y MBP. Para establecer si las vías de señalización MAPK y PI3Kparticipaban en la acción de GH sobre oligodendrogénesis, los inhibidoresde quinasas PD 098059 (10, 30 and 50 µM) y LY294002 (2,5, 5,10 y 20µM)respectivamente. Tanto la expresión basal como la inducida por GH de O4y MBP se abolió totalmente por PD 30µM y LY 2,5µM, y parcialmente porPD 10µM. El tratamiento con GH también incrementó la expresión deMBP en cerebro de ratas viejas. Estos resultados indican que la GH promuevela oligodendrogénesis incrementando el número de pre-OD y la mielinogénesis.También indican que la activación de las vías MAPK y PI3-Kes necesaria para la inducción de OD por GH y así como para el desarrollobasal de los OD. Además, este estudio demuestra que la GH induce el procesode remielinización en el cerebro de la rata vieja

We have previously reported that Growth Hormone (GH) inducesproliferation and differentation of prenatal cerebrocortical cells. The roleof GH in oligodendrocyte (OD) development and myelination is poorlyunderstood. The aim of the study was to investigate whether GH promotesOD maturation and to establish the signaling pathways involved in itsaction. Also, the action of GH on brain remyelination during aging wasstudied. Prenatal rat cerebrocortical cells incubated in DMEM 15% FCSfor 6 h were cultured for 4 days in defined medium with 25ng/ml bFGFand treated for 8 days with 50 ng/ml human recombinat GH. Kinase inhibitors,when used, were added 30 minutes before GH treatment. Adult(3 months) and 27 month-old Wistar rats were treated subcutaneouslywith rhGH (150 µg/12hr). To verify the effect of GH on OD developmentand myelination, pre-OD and mature OD were detected by immunocytochemistryusing O4 and myelin basic protein (MBP) antibodies respectively.MBP was detected in brain by immunohistochemistry. GH increasedthe number of O4 (Control: 100%; GH: 166±5%) and MBPpositive cells. To elucidate whether the MAPK and PI3K signaling pathwayswere involved in GH-induced OD development, the kinase inhibitorsPD 098059 (10, 30 and 50µM) and LY294002 (2.5, 5.10 and 20µM)were used. GH-induced and basal appearance of O4 and MBP positivecells was completely prevented by 30µM PD and 2.5µM LY and partly by10µM PD. GH treatment increased the expression of MBP in the old rats.These results indicate that GH promotes oligodendrogenesis by increasingthe number of pre-OD and myelination. They also show that the activationof MAPK and PI3K pathways is crucial for GH induction of ODas well as basal OD development. In addition, GH promotes remyelinationin the CNS during aging

IGF-I and vasoactive intestinal peptide (VIP) regulate cAMP-response element-binding protein (CREB)-dependent transcription via the mitogen-activated protein kinase (MAPK) pathway in pituitary cells: requirement of Rap1.

In previous studies we demonstrated that vasoactive intestinal peptide (VIP) mediation, and interactions between mitogen-activated protein kinase (MAPK) and cAMP/protein kinase A (PKA) signaling pathways are implicated in insulin-like growth factor I (IGF-I)- and VIP-induced lactotroph proliferation. These facts led us to investigate the intracellular mechanisms involved in IGF-I- and VIP-induced lactotroph proliferation. Exposure of cultured male rat pituitary cells to IGF-I (10(-7) M) or VIP (10(-7) M) stimulated the MAPK cascade. Studies in GH4C1 cells, with an expression vector for Rap1 GTPase-activating protein (Rap1 GAP1), demonstrated reduced VIP-induced MAPK activation, indicating that VIP-dependent activation of the extracellular signal-regulated kinase (ERK) pathway requires PKA-Rap1 signaling. IGF-I induced cAMP-response element (CRE)-binding protein (CREB) phosphorylation through the Ras-MAPK pathway, whereas VIP phosphorylated CREB directly via PKA. The mechanisms that regulate IGF-I-and VIP-CREB-dependent gene transcription were examined using GH4C1 cells transiently transfected with a CRE reporter gene. IGF-I and VIP stimulation of CRE-mediated transcription required activation of both Ras-MAPK and cAMP/PKA signaling. This activation was blocked in the presence of Rap1 GAP1. In summary, we showed that IGF-I and VIP stimulated MAPK activity and the phosphorylation of CREB in pituitary cells. Furthermore, VIP-dependent activation of PKA-Rap1-ERK pathways mediated VIP and IGF-I effects on CREB-dependent transcription in GH4C1 cells. Thus, it is possible that VIP- and IGF-I-induced lactotroph proliferation may involve Rap1.

Directrices para el tratamiento del carcinoma diferenciado de tiroides / Guidelines for the treatment of differentiated thyroid carcinoma

En los últimos 4 años ha cambiado significativamente la estrategia terapéutica y del seguimiento del cáncer diferenciado de tiroides (CDT). Estos cambios se refieren a la estratificación de riesgo, a la intensidad del tratamiento postoperatorio y a los criterios de seguimiento. El uso de tiroglobulina circulante (TG), como marcador tumoral, el uso de tirotropina humana recombinante (TSHrh) y técnicas de localización morfológica o funcional, como la ecografía de alta resolución y la tomografía por emisión de positrones con 18F-fluordesoxiglucosa, son el fundamento de este cambio. En múltiples estudios se ha confirmado que la experiencia del cirujano es importante para la eliminación completa de tejido tiroideo y metástasis locorregionales. Se han establecido nuevos criterios de remisión tumoral basados en TG indetectable, con anticuerpos antitiroglobulina negativos, con tirotropina TSH elevada (> 25 µU/ml). Con estos marcadores y con las exploraciones morfológicas se establecen criterios de remisión, criterios de intervención-actuación y criterios de reintervención quirúrgica. Debido a la mayor sensibilidad de la TG como marcador tumoral se considera actualmente de mínima utilidad el rastreo diagnóstico, tanto para la estratificación de riesgo como para la decisión de actuaciones terapéuticas. Los restos tumorales no tratables quirúrgicamente, ni con dosis ablativas de 131I, pueden tener indicación de tratamiento con radiación externa. Se han consolidado las condiciones para el tratamiento ablativo con radioyodo, tanto en retirada de la tiroxina como mediante tratamiento con TSHrh. También se han establecido las indicaciones y los protocolos diferentes para el uso de TSHrh en el tratamiento y seguimiento del cáncer diferenciado de tiroides. Se ha consolidado el concepto de que en situaciones de bajo riesgo y de remisión, con los marcadores establecidos, sólo es necesario tratamiento sustitutivo con tiroxina, no es imprescindible tratamiento supresor. Es recomendable evitar exceso de aporte de yodo en período próximo a las dosis terapéuticas de radioyodo (AU)

In the last four years the therapeutic approach and follow-up of differentiated thyroid cancer (DTC) has changed substantially. These changes relate to risk stratification, the intensity of postoperative treatment, and follow-up criteria. Underlying this change are the use of the circulating thyroglobulin (TG) as a tumoral marker, the use of recombinant human thyroid-stimulating hormone (rhTSH), and techniques of morphological or functional localization such as high resolution ultrasonography and 18F-fluorodesoxyglucose positron emission tomography (FDG-PET). Numerous studies have confirmed that the experience of the surgeon is important for the complete elimination of thyroid tissue and locoregional metastases. New criteria have been established for tumoral remission based on undetectable TG levels, with negative TG antibodies, and elevated TSH (> 25 µU/ml). These markers and morphological explorations can be used to establish remission criteria. Because of the greater sensitivity of TG as a tumoral marker, diagnostic whole body scan is currently considered of little use in risk stratification or therapeutic decision-making. Tumoral remnants not suitable for surgical treatment or ablation doses of 131I may be suitable for treatment with external radiation. The conditions for ablative radioiodine treatment have been established either in T4 withdrawal or rhTSH treatment. The indications and the various protocols for the use of rhTSH in the treatment and follow-up of DTC have also been defined. It is currently believed that in patients with low risk or in remission, with the established markers, only T4 substitution therapy is required and that suppression treatment is not essential. Excessive iodine intake should be avoided around the time that therapeutic doses of radioiodine are administered (AU)

Differential regulation of gonadotropins and glycoprotein hormone alpha-subunit by IGF-I in anterior pituitary cells from male rats.

IGF-I has been demonstrated to stimulate basal and GnRH-induced gonadotropin release. IGF-I also elicites alpha-subunit secretion in human pituitary tumor cells. The aims of this study were to evaluate both the effect of IGF-I on gonadotropin LH-beta and FSH-beta mRNA levels and glycoprotein alpha-subunit gene expression in cultured rat anterior pituitary cells. The exposure of pituitary cells to recombinant human IGF-I (rhlGF-I; 2 microg/ml) for 72 h markedly stimulated basal LH and FSH release whereas their mRNA levels remained unmodified. IGF-I elicited a-subunit release from pituitary cells (p < 0.01) and augmented its mRNA levels. Exposure to IGF-I consistently reduced GH release from pituitary cells. This study shows that the gonadotropin-releasing effects of IGF-I are not paralleled by changes in their mRNAs whereas IGF-I stimulates not only alpha-subunit release but also its mRNA levels. This study provides the first observation of alpha-subunit regulation by IGF-I in normal pituitary cells, where a differential regulation between release and synthesis for gonadotropin a-and 1-subunits is also shown.

IGF-I inhibits apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway in pituitary cells.

In previous studies we demonstrated that IGF-I induces proliferation of pituitary lactotrophs. In addition to its mitotrophic actions, IGF-I is known to prevent apoptosis induced by diverse stimuli in several cell types. In this study, we investigated the action of IGF-I on pituitary cell survival and the intracellular signaling transduction pathway implicated in this effect. Treatment of cultured male rat pituitary cells with IGF-I (10(-7) M) for 24 h prevented pituitary cell death induced by serum deprivation. The protective effect of IGF-I was blocked by phosphoinositide 3-kinase (PI3-kinase) inhibitor, LY294002, but was unaffected by PD98059, which inhibits MAP/ERK kinase (MEK1). IGF-I activation of PI3-kinase induced the phosphorylation and activation of the serine/threonine kinase Akt. Moreover, IGF-I increased the phosphorylation of the pro-apoptotic factor Bad and the levels of the anti-apoptotic protein Bcl-2 through the PI3-kinase pathway in primary pituitary cells.

A novel peculiar mutation in the sodium/iodide symporter gene in spanish siblings with iodide transport defect.

Previously, we reported two Spanish siblings with congenital hypothyroidism due to total failure of iodide transport. These were the only cases reported to date who received long-term iodide treatment over 10 yr. We examined the sodium/iodide symporter (NIS) gene of these patients. A large deletion was observed by long and accurate PCR using primers derived from introns 2 and 7 of the NIS gene. PCR-direct sequencing revealed a deletion of 6192 bases spanning from exon 3 to intron 7 and an inverted insertion of a 431-base fragment spanning from exon 5 to intron 5 of the NIS gene. The patients were homozygous for the mutation, and their mother was heterozygous. In the mutant, deletion of exons 3-7 was suggested by analysis using programs to predict exon/intron organization, resulting in an in-frame 182-amino acid deletion from Met(142) in the fourth transmembrane domain to Gln(323) in the fourth exoplasmic loop. The mutant showed no iodide uptake activity when transfected into COS-7 cells, confirming that the mutation was the direct cause of the iodide transport defect in these patients. Further, the mutant NIS protein was synthesized, but not properly expressed, on the cell surface, but was mostly accumulated in the cytoplasm, suggesting impaired targeting to the plasma membrane.

Involvement of VIP on BDNF-induced somatostatin gene expression in cultured fetal rat cerebral cortical cells.

Previous studies have shown that BDNF promotes expression of SS. In earlier studies we demonstrated the stimulatory effect of locally produced VIP upon SS secretion. These facts led us to explore the peptidergic action of BDNF on VIP, and to determine if BDNF-induced SS might be mediated by the induction of VIP. Cultured fetal rat cerebrocortical cells were incubated with BDNF (50 ng/ml) and/or VIP (10(-11) M) for 2 and 5 days. In other experiments IgGs from BDNF or VIP antisera were also added. BDNF increased VIP and SS gene expression and peptide production. After 2 days of incubation with both BDNF and VIP the induction of SS mRNA was similar to that obtained with BDNF alone. However when the treatment was extended to 5 days the increase in SS mRNA was higher than that obtained with BDNF alone. This finding suggests the possibility that both factors acted synergistically. To define the potential role of VIP in the response of SS gene expression to BDNF, endogenous VIP was blocked with IgGs from VIP antiserum. Under these experimental conditions BDNF-induced SS decreased. Our study provides the first evidence that BDNF up-regulates VIP gene expression and concentration of the peptide. The involvement of VIP on BDNF-induced SS gene expression is also demonstrated.

Sensitivity to exogenous GH and reversibility of the reduced IGF-I gene expression in aging rats.

BACKGROUND: IGF-I gene expression and IGF-I plasma concentration decline with age. A decreased sensitivity to GH has been suggested to be a contributory mechanism to this, in addition to attenuated GH secretion. OBJECTIVE: This study focuses on the sensitivity to exogenous GH and the reversibility of the reduced IGF-I gene expression in aging male rats. DESIGN: Three groups of male Wistar rats aged 3 months (young adult), 11 months (middle-aged) and 27 months (old), received recombinant human GH (rhGH) (150 microg/12 h s.c.) for seven consecutive days. RESULTS: This rhGH treatment completely reversed plasma immunoreactive IGF-I (IR-IGF-I) and hepatic IGF-I mRNA levels in 11-month-old and 27-month-old animals to the levels of the young group of animals. The sensitivity in the old group (percentage of increment after the same or lower dose of rhGH per body weight) was increased for both parameters; serum IGF-I increment: 15% in 3-month-old, 42.6% in 11-month-old and 119.1% in 27-month-old rats; and hepatic IGF-Ib mRNA increase: 45% in 3-month-old, 27.8% in 11-month-old and 64.3% in 27-month-old rats. IGF binding protein-3 (IGFBP-3) mRNA level in the liver was significantly decreased in the old group and only a partial reversion occurred in this group after rhGH treatment; the percentage of increment was also higher in the old group of rats. In extrahepatic tissues IGF-I mRNA was not significantly different in the kidney and the testis of the three groups, and the rhGH treatment produced a significant and similar increase of IGF-I mRNA level in the kidney of the three groups of rats and in the testis of the 27-month-old animals. The GHr/GHBP mRNA remained unchanged in the liver and in the kidney or the testis of the three groups, and was not influenced by the rhGH treatment. Exogenous rhGH decreased pituitary GH mRNA accumulation in a more intense manner in the old group versus control of each group: young adult, 25%; middle-aged, 41.2%; and old rats, 55%. The action of rhGH on pituitary immunoreactive GH (IR-GH) content was only significantly evident in the young group. CONCLUSIONS: These results establish that exogenous rhGH recovers the attenuated liver IGF-I gene expression and the diminished plasma IR-IGF-I in old rats to the levels of young adult animals. They also indicate that the hepatic and extrahepatic (kidney and testis) sensitivity to one established dose per weight of exogenous rhGH is not altered in old animals, or could be potentially increased in some tissues.

[Acromegaly associated with mitral and tricuspid prolapse. Report of a case]. / Acromegalia asociada a prolapso mitral y tricuspídeo. Descripción de un caso.

A 65 year-old male with severe systolic mitral and tricuspid valve prolapse, associated with long-standing acromegaly is reported. The non published association could be caused by the effect of growth hormone on the valve connective tissue.

Mechanisms of reduced body growth in the pubertal feminized male rat: unbalanced estrogen and androgen action on the somatotropic axis.

It is well known that the sex difference in body growth at puberty is modulated by a complex interplay between sex steroids and somatotropic axis; however, the exact role played by sex steroids remains a matter of controversy. The aim of this study was to assess the mechanisms by which sex steroids regulate body growth during pubertal development. Flutamide, a non-steroid-blocking androgen receptor, was subcutaneously administered to 30-d-old male Wistar rats for 4 wk. The blockade of the androgen receptor led to a marked elevation in serum testosterone and an increment in serum estradiol. Flutamide administration decreased body weight gain, serum IGF-I levels, hepatic IGF-I mRNA, and GH receptor mRNA content. There were no significant changes in serum GH concentration, pituitary GH reserve, and pituitary GH mRNA content. Flutamide lowered hypothalamic somatostatin mRNA content and augmented hypothalamic immunoreactive somatostatin stores, but did not alter hypothalamic immunoreactive GH-releasing factor stores. Our findings indicate that during pubertal development of the male rat, the imbalance between androgen and estrogen actions determines an abnormal somatic growth, which is at least partly exerted through the peripheral or hepatic modification of the somatotropic axis that occurs under the high or exclusive action of estrogens. Potential implication of coincident sex-specific regulated mode of pulsatile GH secretion cannot be excluded from this random serum GH sample study.

Regulation of gonadal and somatotropic axis by chronic intraventricular infusion of insulin-like growth factor 1 antibody at the initiation of puberty in male rats.

There has been increasing experimental evidence to suggest that insulin-like growth factor 1 (IGF-I) may be one of the essential regulators in the reproductive system of the rat. IGF-I is synthesized in the hypothalamus and IGF-I immunoreactivity increases during puberty. Consequently we hypothesized that centrally located IGF-I might contribute to the initiation of puberty. Centrally located IGF-I was immunoneutralized to assess this hypothesis. Male Wistar rats, 28 days old, were infused intracerebroventricularly with specific purified IgGs from rabbit IGF-I antiserum (IGF-I-Ab). The intracerebroventricular administration of IGF-I-Ab resulted in a reduction in testicular weight and consequently in delayed pubertal development. There was also a reduction in serum testosterone, pituitary immunoreactive (IR) luteinizing hormone (LH) and serum IR follicle-stimulating hormone (FSH). The accumulation of betaLH mRNA was not modified, whereas betaFSH mRNA was increased. An increment in the serum growth hormone (GH) levels was also observed. There were no significant alterations in hypothalamic IR growth hormone releasing factor content, although IR somatostatin (SRIH) content was increased by IGF-I-Ab. The body weight gain remained unaltered. As a whole, our study suggests that centrally located IGF-I influences pubertal development, production and release of gonadotropins and supports the finding that endogenous centrally located IGF-I plays a role at the initiation of puberty in the male rat. It also gives support to the physiological role of centrally located IGF-I in the release of GH mediated by hypothalamic SRIH at the initiation of puberty.

Tissue-specific response of IGF-I mRNA expression to obesity-associated GH decline in the male Zucker fatty rat.

Diminished GH secretion is a well known association of obesity. As in obese humans, Zucker fatty rats develop a progressive GH deficiency, present at 6 weeks of age and maximal at 10 to 12 weeks. The aim of this study was to investigate the GH dependence of IGF-I gene expression in liver and extrahepatic tissues of the obese Zucker rat as a model of progressive GH reduction during adult life. Six- and 11-week-old obese Zucker rats and their lean littermates were used to compare body weight, glycemia, insulinemia, serum GH and IGF-I levels and IGF-I mRNA expression in liver, heart, aorta, kidney and skeletal muscle. In comparison with lean controls, obese Zucker rats showed at both ages comparable glycemia, severe hyperinsulinemia (mU/ml, mean+/-s.e.m.; 6 weeks 138+/-10 vs 45+/-6 P<0.001; 11 weeks 147+/-14 vs 46+/-3, P<0.001) and lower GH (ng/ml; 6 weeks 1.7+/-0.9 vs 2.7+/-1.1; 11 weeks 1.5+/-0.9 vs 4.2+/-1.2) in the presence of similar circulating IGF-I levels (ng/ml; 6 weeks 774+/-26 vs 694+/-28; 11 weeks 1439+/-182 vs 1516+/-121). Hepatic IGF-I mRNA expression was already reduced at 6 weeks of age due to a significant decrease in the IGF-Ib transcript compared with lean controls (relative units; IGF-Ia: 99+/-2% vs 100+/-5%; IGF-Ib: 69+/-10% vs 100+/-2%, P<0.05) and this reduction was more marked in 11-week-old animals when both IGF-I transcripts were significantly diminished (relative units; IGF-Ia: 80+/-6% vs 100+/-1%, P<0.05; IGF-Ib: 65+/-5% vs 100+/-2%, P<0.01). Extrahepatic tissues expressed almost exclusively the IGF-Ia transcript, the amount of which relative to controls was: (1) similar at 6 weeks and decreased at 11 weeks in kidney and skeletal muscle extracts (relative units; kidney: 6 weeks 88+/-10% vs 100+/-2%; 11 weeks 76+/-3% vs 100+/-4%, P<0.05; vastus lateralis: 6 weeks 95+/-7% vs 100+/-10%; 11 weeks 59+/-4% vs 100+/-2%, P<0.001); (2) similar at both ages in thoracic aorta (relative units; 6 weeks 121+/-6% vs 105+/-5%; 11 weeks: 91+/-14% vs 100+/-4%); and (3) increased at both ages in left ventricle extracts (relative units; 6 weeks 114+/-2% vs 99+/-9%, P<0. 05; 11 weeks 119+/-7% vs 95+/-3%, P<0.05). -specific dependence of IGF-I mRNA on GH levels during adulthood, reflected by the different behavior of IGF-I expression for each tissue in conditions of progressive decrease of GH levels.

Direct action of serotonin on prolactin, growth hormone, corticotropin and luteinizing hormone release in cocultures of anterior and posterior pituitary lobes: autocrine and/or paracrine action of vasoactive intestinal peptide.

There is extensive evidence that serotonin (5-HT) is implicated in the neuroendocrine control regulating the secretion of several anterior pituitary hormones. It has also been reported that the posterior pituitary is necessary for prolactin (PRL) response to 5-HT as well as to suckling, in which 5-HT implication has been demonstrated. As we have previously shown that vasoactive intestinal peptide (VIP) mediates through an autocrine or paracrine action the PRL release induced by insulin-like growth factor I, thyrotropin-releasing hormone (TRH) and dopamine withdrawal, the aim of the present work was to determine whether 5-HT has a direct action on pituitary secretion and to study the possible role of pituitary VIP in this situation. Cells from the anterior pituitary lobe (AP) were cultured either alone or together with cells from the posterior pituitary lobe (PP). As melanotropes from PP express glucocorticoid receptors in vitro, both AP cultures and cocultures of AP/PP cells were incubated in the presence or absence of corticosterone (0.1 microg/ml), thus designing four experimental conditions. Then both AP and mixed cultures were incubated with 5-HT (100 nM) for 20, 45 and 180. The release of PRL, growth hormone (GH), corticotropin (ACTH) and luteinizing hormone (LH) was stimulated by 5-HT, but only in cocultures of AP/PP cells preincubated with corticosterone, whereas follicle-stimulating hormone and thyroid-stimulating hormone release was not modified. As AP cultures did not show any response to 5-HT, both in the presence or absence of corticosterone, and as melanotropes are the main cellular type present in the PP cultures, we studied the response of alpha-melanocyte-stimulating hormone (alphaMSH) to 5-HT in PP cells cultured with or without corticosterone. Serotonin did not modify alphaMSH release either in the absence or the presence of corticosterone. VIP release was also stimulated by 5-HT in the cocultures, and the time response profile was only similar to that of PRL. In order to study whether pituitary VIP is implicated in 5-HT action, cocultures preincubated with corticosterone were incubated in the presence of 5-HT, a VIP-receptor antagonist (VIP-At) or simultaneously with 5-HT plus VIP-At. PRL response to 5-HT was abolished by the simultaneous presence of VIP-At, whereas GH, ACTH and LH response remained unchanged. These data demostrate that: (1) 5-HT stimulates the secretion of PRL, GH, ACTH, LH and VIP acting directly at pituitary level on PP, probably by releasing an unidentified mediator from melanotropes; (2) glucocorticoids make the response of AP cells to 5-HT possible due to the presence of PP cells in the coculture; (3) PRL response to 5-HT is mediated through an autocrine and/or paracrine action of VIP.

Growth hormone gene expression and secretion in aging rats is age dependent and not age-associated weight increase related.

GH secretion declines with age in rats and humans and a reduction in GH gene expression has been demonstrated in aging rats. GH secretion also diminishes in obesity; thus, the aim of this study was to determine whether GH decrease in aging rats is due to body weight gain or to aging. Three groups of male Wistar rats of different ages were studied (young, 3 months; middle-aged, 11 months; old, 27 months). The middle-aged group was established on a statistical analysis and corresponded to the youngest age at which body weight was not significantly different from the old (27 month) group. Thus, by using this group as control for comparison with animals with the same weight and an older age, the effects due to aging itself could be determined. Body weight (g, mean +/- sD) 3 months: 361 +/- 5.6; 11 months: 713 +/- 39; 27 months: 635 +/- 38. In comparison with 3-month-old rats, the 11-month-old animals showed no difference in pituitary GH messenger RNA (mRNA) accumulation and pituitary and serum IR-GH levels. Similarly IGF-I.a, IGF-I.b mRNA transcripts and IG-FBP-3 mRNA accumulation in the liver showed no significant differences between the two groups. On the contrary, when the 27-month-old rats were compared with the 11-month-old animals, lower levels of pituitary GH mRNA and serum and pituitary IR-GH were found. Pituitary GH mRNA decreased 37.5 +/- 7.7% P < 0.001, pituitary IR-GH content diminished (5.2 +/- 3.4 vs. 55 +/- 10.7 ng/mg of protein, P < 0.001) and serum IR-GH decreased (3.5 +/- 1.8 vs. 12.5 +/- 4.2 ng/ml, P < 0.01). Liver IGF-I.a and IGF-I.b mRNA transcripts accumulation and serum IGF-I were significantly diminished. IGF-I.b mRNA accumulation decreased 35.8 +/- 1.2% P < 0.05 and IGF-I.a 36 +/- 5.6% P < 0.05; serum IR-IGF-I levels diminished (759 +/- 152 vs. 1327 +/- 67 ng/ml, P < 0.05). Liver IGFBP-3 mRNA accumulation decreased 79 +/- 4.2% P < 0.001. These results indicate that the decrease in GH gene expression and secretion, as well as the expression of genes induced by GH such as IGF-I and IGFBP-3, is due to aging and not to the increase in body weight that takes place with aging.

Regulation of growth hormone (GH) gene expression and secretion during pregnancy and lactation in the rat: role of insulin-like growth factor-I, somatostatin, and GH-releasing hormone.

GH appears to play an important metabolic role during late pregnancy and in lactation maintenance. In this study, pregnant (days 8, 15, and 20 of gestation) and postpartum (days 3 and 8 postpartum, including lactating and nonlactating dams) Wistar rats were used to investigate pituitary GH gene expression and hormone secretion, and the potential alterations of the major signals regulating GH secretion and action [somatostatin (SS) and GH-releasing hormone (GHRH), GH receptor (GH-R), and insulin-like growth factor-I (IGF-I)]. GH and SS messenger RNA (mRNA) were quantitated by Northern blot, and both IGF-I and GH-R mRNA were analyzed by the ribonuclease protection assay technique. Pituitary IR-GH content and GH mRNA increased at midpregnancy. IR-GH content was decreased in lactating rats. Plasma GH levels progressively increased during pregnancy, whereas no significant alterations were shown during lactation. Elevated GH levels persisted during lactation. Levels at this time were higher in nonsuckling compared with suckling dams. Liver GH-R mRNA progressively decreased during pregnancy, but it remained unchanged during lactation. Plasma IGF-I and liver IR-IGF-I constantly decreased during pregnancy, and no significant modifications were seen either in suckling or in nonsuckling animals. IGF-I mRNA accumulation in the liver decreased during pregnancy. After delivery, a progressive decrease of liver IGF-I mRNA occurred. At the hypothalamic level, a progressive increase in the IR-SS content was found during pregnancy, with no SS mRNA modification. After delivery, a higher hypothalamic IR-SS content was found in lactating than in nonlactating rats, with no changes in SS mRNA levels. Hypothalamic IR-IGF-I also showed a progressive increase during pregnancy with no significant alterations during lactation. Hypothalamic IR-GHRH presented a nonsignificant mild increase during pregnancy with no modifications during lactation. In the pituitary, IR-IGF-I content progressively increased during gestation, reaching its highest concentration at day 20. During lactation, pituitary IGF-I did not change. In summary, our data show that the mechanisms of the increase in plasma GH levels occurring during pregnancy include an increase in GH gene expression in the pituitary, a decrease in SS secretion from the hypothalamus, an increase in IR-IGF-I content in the hypothalamus and in the pituitary, and a significant decrease in circulating IGF-I. Plasma and liver IR-IGF-I and IGF-I mRNA in the liver decreased throughout gestation due to a lower GH-R gene expression in the liver. This state of GH resistance with a higher GH/IGF-I ratio could be important in providing supplementary nutrients to the fetus. During lactation, GH and its regulatory machinery did not show important modifications.

Basal and growth hormone-induced hepatic messenger ribonucleic acid expression of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 is independent of hyperinsulinemia and increased energy status in the genetically obese Zucker rat.

Genetically obese Zucker rats, like obese humans, have normal or elevated circulating insulin-like growth factor-I (IGF-I) levels in the presence of low GH secretion. Hyperinsulinemia, increased energy status, or other nutritional factors associated with obesity could be responsible for these findings directly by increasing hepatic IGF-I production at the transcriptional or posttranscriptional level. Alternatively, circulating IGF-I could be modulated indirectly by affecting its binding proteins. To further elucidate this point, we quantitated hepatic IGF-I, IGF-binding protein-3 (IGFBP-3), and GH receptor messenger RNAs (mRNAs) expression in obese Zucker rats under different serum GH and insulin conditions using lean rats as controls. Eleven-week-old male rats were studied basally (intact) or after hypophysectomy (hx) at 9 weeks. In each condition, animals were killed before or 6 h after one dose of recombinant human GH (1.5 micrograms/g body weight ip). At this time, in addition to the mRNA expression of the above-mentioned genes, body weight, glycemia, insulinemia, serum GH (rat and human), and serum IGF-I levels were determined. Obese Zucker rats were significantly heavier than controls in all the conditions studied and did not show differences in glycemia. Severely hyperinsulinemic intact obese rats (146.9 +/- 14 vs. 46.3 +/- 3 microU/ml, P < 0.001) showed compared with intact lean rats significantly lower serum GH (2.39 +/- 0.9 vs. 4.98 +/- 0.68 ng/ml, P < 0.01), decreased hepatic IGF-I mRNA and IGFBP-3 mRNA accumulation (IGF-Ia: 79 +/- 5.9% vs. 100 +/- 0.9%, P < 0.05; IGF-Ib: 67 +/- 5.5% vs. 100.1 +/- 1.9%, P < 0.001; IGFBP-3: 54.7 +/- 2.75% vs. 100.5 +/- 1.55%, P < 0.001), and similar circulating IGF-I levels (1439 +/- 182 vs. 1516 +/- 121 ng/ml). Under comparable serum GH levels in GH-treated intact, hx, and GH-treated hx animals, hyperinsulinemia and/or increased body weight present in obese rats were not associated with increased hepatic IGF-I and IGFBP-3 mRNA amount. No differences in GH receptor/GH-binding protein mRNAs were found in any experimental condition. These results suggest that in vivo the imbalance of the serum GH/IGF-I axis present in obesity is primarily due to events distal to the hepatic IGF-I and IGFBP-3 mRNAs expression, which is tightly correlated to GH levels.

Growth hormone induces somatostatin and insulin-like growth factor I gene expression in the cerebral hemispheres of aging rats.

The neuropeptide somatostatin (SS) plays a role as a modulator of cognitive functions and as a potential tropic factor in the central nervous system. A reduction in SS levels has been demonstrated in the aging brain and in dementia. In addition, insulin-like growth factor I (IGF-I) acts as a paracrine factor in multiple GH actions and is also found in the cerebral hemispheres, where it exerts neurotropic effects. We used aging rats as an in vivo model of GH deficiency to study the possible participation of exogenous GH in the modulation of the cerebral hemispheric SS and IGF-I. Two sets of experiments were carried out. In the first set, the age-related patterns of GH, IGF-I, and SS in the serum, pituitary, and cerebral hemispheres were established. In the second experimental set, 90-day-old (adult) and 2-yr-old (aging) male rats received recombinant human GH (200 micrograms/ sc-day) or vehicle for 7 consecutive days. The serum levels of rat GH and IGF-I as well as pituitary GH messenger RNA decreased in 2-yr-old rats compared with those in adult rats. After GH treatment, pituitary GH messenger RNA levels decreased markedly in the 90-day-old and 2-yr-old rats. Serum immunoreactive GH decreased in the adult animals, whereas it remained unaffected in the aging ones, whereas serum IGF-I levels were not altered by GH treatment in either group. Immunoreactive levels and messenger RNA of both SS and IGF-I were low in the cerebral hemispheres of aging rats, but were restored to the levels found in adult rats after GH treatment. As treatment did not induce changes in the serum IGF-I levels, these results provide evidence of a stimulatory action of peripherally administered GH on the regulation of SS and IGF-I genes in the aging rat in the central nervous system. These data also show a new target action for GH and could provide a molecular basis for the improvement of some symptoms of GH deficiency that occurs after recombinant human GH treatment.

Autoparacrine action of vasoactive intestinal peptide on dopaminergic control of prolactin secretion.

We have previously reported that pituitary vasoactive intestinal peptide (VIP) mediates through autoparacrine mechanisms insulin-like growth factor-I and TRH-stimulated PRL release. In the present study, we have investigated whether VIP might also be implicated in the PRL increase that follows dopamine (DA) withdrawal. The experiments were carried out in defined medium supplemented or not with T3, as in a previous study we had found that PRL release increases under low T3 culture conditions due to mediation by concomittant stimulus of VIP. Anterior pituitary (AP) cells from adult male rats were incubated for 1 h in the presence of DA (10 microM), a VIP-receptor antagonist (VIP-At) (200 nM), or DA plus VIP-At. Then media were removed and the cells were washed with PBS and reincubated under the same conditions but without the addition of DA. In the first incubation, DA inhibited PRL release by 80%, and VIP release by 20% in both T3-free and T3 medium. In the second period of incubation, PRL and VIP release were increased in AP cells previously exposed to DA. These effects were greater when the cells were cultured in T3-free medium than in T3-medium (225% and 150%, respectively for PRL release; and 155 and 135%, respectively for VIP release). PRL response to DA withdrawal was inhibited by the simultaneous presence of VIP-At. This inhibition was again greater when the cells were incubated in medium supplemented with T2. Thus, the stimulus of DA withdrawal was inhibited by 88% in T2-free medium and by 37% in T3-medium. To investigate whether pituitary VIP messenger RNA (mRNA) expression is under dopaminergic control, AP cells were incubated in the presence or absence of bromocriptine (BC) (10 nM) for 2 and 24 h. After 24 h of incubation, BC decreased mRNA levels of PRL and of the two transcripts which VIP expresses in AP. As with DA, BC also inhibited PRL and VIP release both at 2 and 24 h. These data demonstrate that VIP, at least partially, mediates DA withdrawal-induced PRL release through an autoparacrine action. They also suggest that simultaneous inhibition of pituitary VIP mRNA expression and VIP release may be a necessary mechanism for the dopaminergic inhibition of PRL mRNA expression and PRL release.

Prolactin gene expression and secretion during pregnancy and lactation in the rat: role of dopamine and vasoactive intestinal peptide.

It is known that dopamine (DA) is the major PRL-inhibiting factor, and vasoactive intestinal peptide (VIP) is one of the most potent and physiological PRL-releasing factors. We have investigated the implication of DA and VIP in PRL gene expression and peptide secretion regulation during the physiological hyperprolactinemic states of pregnancy and lactation. Pregnant rats were studied on days 8, 15, and 20 of pregnancy. Lactating rats suckled by eight pups were studied on days 3 and 8 of postpartum, and nonsuckling postpartum rats were used as controls. Plasma estradiol, progesterone, and PRL were measured by RIA, as well as pituitary immunoreactive (IR-) PRL, pituitary IR-VIP, and hypothalamic IR-VIP. DA was studied by measuring changes in gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. TH, PRL, and VIP messenger RNA (mRNA) were assessed by Northern blot hybridization. The results showed very high plasma PRL levels in early pregnancy and during lactation, whereas plasma PRL concentrations were normalized at the end of gestation and in nonsuckling control rats. The physiological hyperprolactinemia of both early pregnancy and lactation correlated with higher pituitary PRL mRNA levels and lower pituitary IR-PRL content. Moreover, hypothalamic TH mRNA levels were lower in early pregnancy and lactation than at the end of gestation and in nonsuckling rats, respectively. The hypothalamic IR-VIP content was lower on day 8 of pregnancy than on days 15 and 20. However, VIP gene expression in the hypothalamus did not change throughout pregnancy. During lactation, neither hypothalamic IR-VIP content nor VIP mRNA was significantly altered. In the pituitary, IR-VIP content did not significantly change, and VIP mRNA levels were higher on day 15 of pregnancy than on the other days. During lactation, the pituitary IR-VIP content was very low on day 8 compared with those on day 3 of lactation and in nonsuckling control rats. VIP mRNA 1.0-kilobase transcript levels were higher in the lactating rats than in the control animals. These data show that both early pregnancy and lactation are physiological hyperprolactinemic states in which increased PRL mRNA accumulation coincides with decreased IR-PRL content in the pituitary and higher plasma IR-PRL, indicating regulation at the gene expression level and of PRL secretion. Low TH gene expression also occurs during hyperprolactinemia, suggesting that the diminution of DA activity that occurs during early pregnancy and lactation might be the major regulator of PRL alterations. If hypothalamic VIP plays a role as a neuroendocrine PRL-releasing factor during pregnancy and lactation, this may occur at the secretory level, as suggested by the alterations in IR-VIP, with no modifications in VIP mRNA accumulation, in the hypothalamus. Pituitary VIP does not seem to be a major regulator of PRL secretion during pregnancy, whereas during lactation, it regulates PRL secretion in a paracrine and/or autocrine manner.