ABSTRACT

The aim of this study was to investigate the role of thyroid hormones and glucocorticoids on GH secretion. Secretion of GH in response to GH-releasing hormone (GHRH) (5 μg/kg) was markedly (P < 0·001) decreased in hypothyroid rats in vivo (peak GH responses to GHRH, 635 ± 88 μg/l in euthyroid rats vs 46 ±15 μg/l in hypothyroid rats). Following treatment with tri-iodothyronine (T₃; 20 μg/day s.c. daily for 2 weeks) or cortisol (100 pg/day s.c. for 2 weeks) or T₃ plus cortisol, a marked (P <0·01) increase in GH responses to GHRH was observed in hypothyroid rats (peak GH responses, 326 ±29 μg/l after T₃ vs 133+19 μg/l after cortisol vs 283 ± 35 μg/l after cortisol plus T₃). In contrast, none of these treatments modified GH responses to GHRH in euthyroid animals. Hypothyroidism was also associated with impaired GH responses to the GH secretagogue, Hisd-Trp-Ala-Trp-d-Phe-Lys-NH₂ (GHRP-6). Secretion of GH in response to GHRP-6 in vivo was reduced (P <0·01) in hypothyroid rats (peak GH responses, 508 ± 177 μg/l in euthyroid rats vs 203 ± 15 μg/l in hypothyroid rats). In-vitro studies carried out using monolayer cultures of rat anterior pituitary cells derived from euthyroid and hypothyroid rats showed a marked impairment of somatotroph responsiveness to both GHRP-6 and somatostatin in cultures derived from hypothyroid rats. In summary, our data suggest that thyroid hormones and glucocorticoids influence GH secretion by modulating somatotroph responsiveness to different GH secretagogues.

Journal of Endocrinology (1989) 121, 31–36

ABSTRACT

His-d-Trp-Ala-Trp-d-Phe-Lys-NH₂ (GHRP-6) is a synthetic peptide unrelated to any known hypothalamic-releasing hormone including growth hormone-releasing hormone (GHRH). Interestingly, this peptide induces a dose-related increase in plasma GH levels in all species tested so far. The aim of this study was to investigate the action of GHRP-6 alone or in combination with GHRH on GH release in dogs. In addition, the activation or blockade of endogenous cholinergic tone and α-1 adrenoceptors on GHRP-6-stimulated GH secretion was assessed.

In adult Beagle dogs (n = 10), GHRP-6 (90 μg i.v.) increased basal GH levels from 2·6 ± 1·5 to 14·4 ± 3·1 μg/l (mean ± s.e.m.) after 15 min. GHRH (50 μg i.v.) induced a GH peak of 9·7 ± 2·2 μg/l at 15 min. The combined administration of GHRP-6 and GHRH strikingly potentiated canine GH release with a peak of 54 ± 9·0 μg/l (P <0·01). Pretreatment with the cholinergic agonist pyridostigmine (30 mg per os) increased GHRP-6-stimulated GH secretion (37·9 ± 10·1 μg/l P <0·05), while the muscarinic blocker atropine (100 μg i.v.) completely abolished (GH peak lower than 2 μg/l) the stimulatory action of GHRP-6. On the other hand, administration of the α-2 adrenergic agonist clonidine (4 pg/kg i.v.) increased basal plasma GH levels without affecting GH responses to GHRP-6. Finally, while the α-1 adrenergic agonist methoxamine (5 mg i.v.) did not significantly increase GH responses to GHRP-6, administration of the α-1 adrenoceptor antagonist prazosin (20 mg i.v.) reduced GHRP-6-induced GH secretion (area under curve, 206 ± 39 vs 557 ± 172, P <0·05).

In summary, the synergistic effect of the combined administration of maximal doses of GHRP-6 and GHRH suggests that these two peptides act through different mechanisms. The finding that cholinergic drugs were able to modulate the GH secretion elicited by GHRP-6 argues against the hypothesis that such a peptide acts by influencing hypothalamic somatostatin release and suggests that it acts directly at the pituitary level. Finally, the unexpected lack of effect of clonidine and the inhibitory effect of prazosin on GHRP-6-induced GH secretion suggests that the role of α-adrenergic pathways in GH secretion is more complex than previously thought.

Journal of Endocrinology (1993) 138, 211–218

ABSTRACT

We have tested the hypothesis that α-adrenergic drive is involved in the nocturnal increase in TSH in man. Seven mildly hypothyroid women (basal TSH levels 5·0–11·0 mU/1), aged 38–60 years, and nine euthyroid women, aged 27–60 years, were studied. Subjects underwent α-adrenergic blockade by infusion of thymoxamine (210 μg/min from 19.00 to 24.00 h); the same women were used as controls, with saline infused on different nights. Subjects were not allowed to sleep during the study period.

A clear evening rise in basal TSH levels was apparent in both normal subjects and patients. Although overall secretion of TSH was slightly decreased in normal subjects (mean ± s.e.m. area under the curve, 29·93 ± 0·96 vs 30·71 ±mU/1 per h; P<0·05), thymoxamine infusion did not produce any major alteration in the gradual rise in TSH levels during the evening (incremental change above baseline +0·96± 0·21 during control infusion and + 0·97 ± 0·27 mU/1 during thymoxamine infusion). In mildly hypothyroid patients the TSH changes were exaggerated and α-adrenergic blockade caused a reduction in basal TSH levels and a delayed rise in TSH (incremental change above baseline +2·93 ± 1·42 during control infusion and +2·26 ± 0·73 mU/1 during thymoxamine infusion; P < 0·02). Overall TSH secretion was significantly decreased by thymoxamine (mean ± s.e.m. area 106 ± 2·45 mU/1 per h vs 123·32 ± 3·68 in the control study; P<0·0001). As expected, no circadian change was observed in basal prolactin levels in either controls or patients.

Although α-adrenergic pathways may play a role in modulating the nocturnal increase in basal TSH levels, our data suggest that the evening rise in TSH is not a consequence of a primary increase in α-adrenergic drive. The increased TSH changes of mildly hypothyroid patients may, however, be sustained by increased central α-adrenergic stimulation of the TSH secretion.

J. Endocr. (1987) 115, 187–191

ABSTRACT

Recent data suggest that the response of GH to GH-releasing factor (GRF) is reduced following prior exposure to high concentrations of GRF. However, it is unknown whether this is due to alterations in GRF receptors, adenylate cyclase activity or the size of a GRF-releasable storage pool of GH. In order to clarify these questions we have compared the effects of pretreatment with GRF (10 nmol/l every 2 h for 12 h) with those of pretreatment with somatostatin (SRIF; 1 μmol/l), forskolin (10 μmol/l) and GRF plus SRIF (10 nmol/l and 1 μmol/l added together) on the subsequent responses of GH to GRF (1 pmol/l–10 nmol/l), cholera toxin (10 nmol/l), 3-isobutyl-1-methylxanthine (IBMX) (100 μmol/l) and forskolin (10 μmol/l). Experiments were performed on 4-day monolayer cultures of rat anterior pituitary cells. The cells were pretreated with test substances every 2 h for 12 h and incubated with GRF or forskolin for 3 h.

Per cent maximal (B_max) GH responses to GRF (10 nmol/l) were reduced after pretreatment with both GRF (control, 173% of basal; GRF, 25% of basal; P < 0·001) and forskolin (98% of basal; P < 0·001), but were increased after pretreatment with SRIF (246% of basal; P < 0·02). However, GH responses after pretreatment with GRF plus SRIF were not significantly different from those of the control. The dose giving 50% maximum stimulation (ED₅₀) of GRF action was unaltered by SRIF pretreatment (control 48·5 ± 9·2 (s.e.m.) pmol/l vs 140 ± 38 pmol/l; P > 0·05) or by forskolin pretreatment (140 ± 70 pmol/l; P > 0·05), but was increased by GRF pretreatment (900 ± 230 pmol/l; P < 0·05) and GRF plus SRIF pretreatment (1010 ± 730 pmol/l; P < 0·01). Growth hormone responses to forskolin (32% of basal after GRF pretreatment vs 121% of basal in the control saline-treated cells; P < 0·01), cholera toxin (40% of basal after GRF pretreatment vs 106% of basal in the control cells; P < 0·025) and IBMX (195% of basal in the GRF-pretreated cells vs 133% of basal in the control cells; P < 0·025) were also decreased after GRF pretreatment in comparison with controls. Responses of cAMP to 10 μmol forskolin/l were not different from controls after pretreatment with either GRF, SRIF or GRF plus SRIF. However, cAMP responses to 10 nmol GRF/l were reduced after pretreatment with GRF, SRIF, GRF plus SRIF or forskolin.

Reduction of B_max GH responses to GRF, cholera toxin, IBMX and forskolin by GRF pretreatment, and the reversal of this action by combined SRIF plus GRF pretreatment, suggests that depletion of the GRF-sensitive releasable pool of GH may contribute to the desensitization phenomenon. In addition, however, the increase in ED₅₀ of GRF following GRF or GRF plus SRIF pretreatment, as compared with control or SRIF pretreatment alone, together with decreased cAMP responses to GRF after GRF plus SRIF pretreatment, supports the view that uncoupling of the regulatory protein of adenylate cyclase, N_s, also plays a role in this phenomenon.

J. Endocr. (1988) 116, 185–190

Leptin is a circulating hormone secreted by adipose tIssue which acts as a signal to the central nervous system where it regulates energy homeostasis and neuroendocrine processes. Although leptin modulates the secretion of several pituitary hormones, no information is available regarding a direct action of pituitary products on leptin release. However, it has been pointed out that leptin and TSH have a coordinated pulsatility in plasma. In order to test a direct action of TSH on in vitro leptin secretion, a systematic study of organ cultures of human omental adipose tIssue was performed in samples obtained at surgery from 34 patients of both sexes during elective abdominal surgery. TSH powerfully stimulated leptin secretion by human adipose tIssue in vitro. In contrast, prolactin, ACTH, FSH and LH were devoid of action. These results suggest that leptin and the thyroid axis maintain a complex and dual relationship and open the possibility that plasmatic changes in TSH may contribute to the regulation of leptin pulses.

Leptin, the product of the ob gene, is secreted into the circulation by white adipose tissue; its major role being to participate in the regulation of energy homeostasis. Plasma leptin levels are mainly determined by the relative adiposity of the subject; however, the great dispersion of values for any given body mass index and the noteworthy gender-based differences indicate that other factors are operating. Steroid hormones actively participate in the regulation of leptin secretion; however, non-steroid nuclear hormones have either not been studied or have provided contradictory results. In order to understand the role of hormones of the non-steroid superfamily such as 3,5,3'-tri-iodothyronine (T(3)), vitamin D(3) and retinoic acid (RA) in the control of leptin secretion, in the present work doses of 10(-9), 10(-8) and 10(-7) M of these compounds have been studied on in vitro leptin secretion. The organ culture was performed with omental adipose tissue samples from healthy donors (n=28). T(3) was devoid of effect at any dose studied, while an inhibition of leptin secretion was observed with 9-cis-RA (slight) and all-trans-RA (potent). Interestingly, vitamin D(3) exerted a powerfully inhibitory role at the doses studied, and its action was synergistic with all-trans-RA. In conclusion, in vitro leptin secretion by human adipose tissue is negatively controlled by either RA or vitamin D(3). The clinical significance of leptin regulation by this superfamily of nuclear receptors remains to be ascertained.

ABSTRACT

Effects of thyroid status on brain catecholamine turnover in adult rats were investigated using a steady-state method. Rats were treated for 3 weeks with s.c. injections of l-thyroxine (0·4 mg/kg), aminotriazole in drinking water (0·1%, w/v) or vehicle. After 2 weeks of treatment rats were implanted chronically with lateral intracerebroventricular (i.c.v.) cannulae. They were injected i.c.v. with [³H]tyrosine 1 week later. Catecholamine and tyrosine content and specific activity were measured in mediobasal hypothalamus, anterior hypothalamus and striatum, using high-performance liquid chromatography with electrochemical detection. Thyroxine treatment resulted in a significant increase in noradrenaline and dopamine synthesis localized to the mediobasal hypothalamus. Conversely, aminotriazole treatment resulted in a significant decrease in noradrenaline synthesis localized to the mediobasal hypothalamus. The localization of these changes in catecholamine turnover to the mediobasal hypothalamus suggests that they may be specific functional effects which are of importance in the overall integrated control of thyroid function.

J. Endocr. (1986) 111, 383–389

ABSTRACT

We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 μmol/l (increase over basal, 118 ±16·5 (s.e.m.) fmol TRH; P <0·001 vs basal). This effect was mimicked by the specific D₂-agonist drugs bromocriptine (0·1 μmol/l) and LY 171555 (0·1 μmol/l) (increase over basal values, 137·5±13·75 fmol and 158·6± 10·7 fmol respectively; P <0·001 vs basal), but not by the D₁-agonist SKF 38393A. The stimulatory effect of dopamine (1 μmol/l) was blocked in a stereospecific manner by the active (d) but not by the inactive (l) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D₂-antagonist domperidone (0·01 μmol/l) whereas the D₁-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0·01 μmol/l) of this D₁ -antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D₂-receptors.

J. Endocr. (1987) 115, 419–424

Leptin, the adipocyte-produced hormone that plays a key role in body weight homeostasis, has recently been found to be involved in the regulation of the hypothalamic-pituitary-adrenal axis. Moreover, reciprocal interactions between leptin and glucocorticoids have been described. In the present communication, two different strategies were undertaken to explore the mode of action of leptin in the direct control of rat adrenal function. First, a synthetic peptide approach demonstrated that the inhibitory effect of leptin on basal and ACTH-stimulated corticosterone secretion in vitro is, at least partially, mapped to a domain of the native protein between amino acids 116 and 130, i.e. an area of the molecule also relevant in terms of regulation of food intake and endocrine control. Secondly, semi-quantitative RT-PCR analysis indicated a complex pattern of adrenal leptin receptor (Ob-R) mRNA expression, with predominant expression of the Ob-Ra and Ob-Rb isoforms, as well as moderate levels of the Ob-Rc and Ob-Rf variants, whereas negligible signals for the Ob-Re isoform were detected. Interestingly, such an expression pattern appeared hormonally regulated as exposure to human recombinant leptin (10(-7 )M) or ACTH (10(-7 )M) significantly decreased Ob-R isoform mRNA expression. Indeed, dose-dependent ligand-induced Ob-Ra and Ob-Rb mRNA down-regulation was further confirmed by adrenal stimulation with increasing concentrations (10(-9)-10(-5 )M) of the active leptin fragment, leptin 116-130 amide. Overall, our results provide evidence for a novel regulatory step at the level of Ob-R mRNA expression in the interplay between ACTH and leptin for the tuning of rat adrenal corticosterone secretion. Furthermore, our data showing down-regulation of Ob-R mRNA expression by its cognate ligand may well be relevant to leptin physiology and its alteration in various disease states.

The use of GH to treat heart failure has received considerable attention in recent years. Although the mechanisms of its beneficial effects are unknown, it has been implicated in the regulation of apoptosis in several cell types, and cardiomyocyte apoptosis is known to occur in heart failure. We therefore decided to investigate whether GH protects cardiomyocytes from apoptosis. Preliminary experiments confirmed the expression of the GH receptor (GHR) gene in primary cultures of neonatal rat cardiomyocytes (PC), the specific binding of GH by HL-1 cardiomyocytes, and the GH-induced activation of GHR and its classical downstream effectors in the latter. That GH prevented the apoptosis of PC cells deprived of serum for 48 h was shown by DNA electrophoresis and by Hoechst staining assays in which GH reduced the percentage of cells undergoing apoptosis. Similarly, the TUNEL-evaluated pro-apoptotic effect of cytosine arabinoside (AraC) on HL-1 cells was almost totally prevented by pre-treatment with GH. Fluorescence-activated cell sorter (FACS) analysis showed apoptosis in 9.7% of HL-1 cells growing in normal medium, 21.1% of those treated with AraC and 13.9% of those treated with AraC+GH, and that GH increased the percentage of AraC-treated cells in the S/G(2)/M phase from 36.9% to 52.8%. GH did not modify IGF-I mRNA levels or IGF-I secretion in HL-1 cells treated with AraC, and the protection afforded by GH against AraC-induced apoptosis in HL-1 cells was not affected by the presence of anti-IGF-I antibodies, but was largely abolished by the calcineurin-inhibiting combination cyclosporin+FK506. GH also reduced AraC-induced phosphorylation of mitogen-activated protein kinase p38 (MAPK p38) in HL-1 cells. In summary, GH protects PC and HL-1 cells from apoptosis. This effect is not mediated by IGF-I and may involve MAPK p38 as well as calcineurin.