Role of growth hormone in regulating T-dependent immune events in aged, nude, and transgenic rodents

Growth hormone (GH) appears to play a major role in a reciprocal axis that has been postulated between the thymus and pituitary glands. Our previous studies showed that thymic structure, as well as T-cell proliferation and IL-2 synthesis, could be restored in aged female Wistar-Furth rats by the implantation of GH3 pituitary adenoma cells. These cells secrete GH and some prolactin. We have now used three different approaches to determine whether GH affects a variety of immune events in vivo in both old and young rodents, and whether GH3 cells can directly affect progenitor T-cells in nude rats that congenitally lack a thymus gland. To test the effects of GH in aged rats, 750 micrograms of pituitary-derived ovine GH was injected 2 x daily into 26-month-old Fischer 344 rats for 5 weeks. This approach demonstrated that GH augments splenocyte proliferation to T-cell lectins as well as natural killer (NK) activity at low effector:target ratios even though morphologic characteristics of the thymus were not altered. To assess the effect of GH in young rodents, mice were studied that were transgenic for the rat metallothionein-GH gene. Histologic evaluation of thymus glands revealed that the amount of adipose tissue and the number of epithelial cells and Hassall's corpuscles are augmented in transgenic mice. Splenocyte proliferation at suboptimal mitogen doses is greater in transgenic than in control littermate mice, but neither IL-2 synthesis nor antibody synthesis to sheep erythrocytes is affected. The role of pituitary hormones on progenitor T-cells was then explored by implanting GH3 cells into Rowett nude rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pregnancy-induced increase in baseline circulating growth hormone in rats is not induced by ghrelin

The elevation in baseline circulating growth hormone (GH) that occurs in pregnant rats is thought to arise from increased pituitary GH secretion, but the underlying mechanism remains unclear. Distribution, Fourier and algorithmic analyses confirmed that the pregnancy-induced increase in circulating GH in 3-week pregnant rats was due to a 13-fold increase in baseline circulating GH (P < 0.01), without any significant alteration in the parameters of episodic secretion. Electron microscopy revealed that pregnancy resulted in a reduction in the proportion of mammosomatotrophs (P < 0.01) and an increase in type II lactotrophs (P < 0.05), without any significant change in the somatotroph population. However, the density of the secretory granules in somatotrophs from 3-week pregnant rats was reduced (P < 0.05), and their distribution markedly polarised; the granules being grouped nearest the vasculature. Pituitary GH content was not increased, but steady-state GH mRNA levels declined progressively during pregnancy (P < 0.05). In situ hybridisation revealed that pregnancy was accompanied by a suppression of GH-releasing hormone mRNA expression in the arcuate nuclei (P < 0.05) and enhanced somatostatin mRNA expression in the periventricular nuclei (P < 0.05), an expression pattern normally associated with increased GH feedback. Although gastric ghrelin mRNA expression was elevated by 50% in 3-week pregnant rats (P < 0.01), circulating ghrelin, GH-secretagogue receptor mRNA expression and the GH response to a bolus i.v. injection of exogenous ghrelin were all largely unaffected during pregnancy. Although trace amounts of 'pituitary' GH could be detected in the placenta with radioimmunoassay, significant GH-immunoreactivity could not be observed by immunohistochemistry, indicating that rat placenta itself does not produce 'pituitary' GH. Although not excluding the possibility that the pregnancy-associated elevation in baseline circulating GH could arise from alternative extra-pituitary sources (e.g. the ovary), our data indicate that this phenomenon is most likely to result from a direct alteration of somatotroph function.     

The inhibition of inducible nitric oxide synthase reverts arthritic-induced decrease in pituitary growth hormone mRNA but not in liver insulin-like growth factor I mRNA expression

Experimental arthritis induced by Freund-adjuvant administration is a model of chronic inflammation and rheumatoid arthritis associated with a decrease in pituitary growth hormone (GH) and hepatic insulin-like growth factor I (IGF-I) gene expression. Excessive nitric oxide (NO) synthesis by inducible NO synthase (iNOS) has been implicated in the pathogenesis of inflammatory illness. Moreover, NO participates in the regulation of GH secretion at both the hypothalamus and the pituitary. We have examined the role of iNOS activation in producing the changes in the GH-IGF-I axis in arthritic rats. Adult male Wistar rats received aminoguanidine or vehicle from day 20, after adjuvant or vehicle injection, until day 28. Two hours and 30 min after the last aminoguanidine injection, all rats were killed by decapitation. Arthritis increased hypothalamic expression of somatostatin mRNA while it decreased pituitary GH mRNA expression, and both effects were prevented by aminoguanidine administration. In arthritic rats, the parallel decrease in serum IGF-I, and in hepatic IGF-I content and mRNA expression, correlates with the decrease in circulating GH concentrations. Aminoguanidine administration to arthritic rats did not modify either serum GH or serum IGF-I concentrations, or hepatic IGF-I mRNA expression. However, aminoguanidine administration to control rats resulted in a decrease in serum GH concentrations and in a decrease in both hepatic IGF-I mRNA expression and serum IGF-I concentrations. These data suggest that NO mediates the arthritis-induced decrease in GH mRNA expression by acting at a hypothalamic level, but it is not involved in the decrease in hepatic IGF-I mRNA expression.     

Expression of ghrelin receptor mRNA in the rat and the mouse brain

Ghrelin is a hormone that stimulates growth hormone secretion and signals energy insufficiency via interaction with its receptor, the growth hormone secretagogue receptor (GHSR). The GHSR is located in both the central nervous system and the periphery. Its distribution in the CNS, as assessed by in situ hybridization histochemistry (ISHH), has been described previously in a few mammalian species, although these studies were limited by either the detail provided or the extent of the regions examined. In the present study, we systematically examined the distribution of GHSR mRNA in the adult rat and mouse brains and cervical spinal cords by using ISHH with novel cRNA probes specific for the mRNA encoding functional GHSR (the type 1a variant). We confirmed GHSR mRNA expression in several hypothalamic nuclei, many of which have long been recognized as playing roles in body weight and food intake. GHSR also was found in several other regions previously unknown to express GHSR mRNA, including many parasympathetic preganglionic neurons. Additionally, we found GHSR mRNA within all three components of the dorsal vagal complex, including the area postrema, the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus. Finally, we examined the coexpression of GHSR with tyrosine hydroxylase and cholecystokinin and demonstrate a high degree of GHSR mRNA expression within dopaminergic, cholecystokinin-containing neurons of the substantia nigra and ventral tegmental area.     

Chronic changes in thyroid hormones do not affect brain adenosine receptors

Stein Murray B., Mike Clark and Suzanne M. Delaney: Chronic changes in thyroid hormones do not affect brain adenosine receptors. Prog Neuro-Psychopharmacol & Biol Psychiat. 1993, 17(6): 1037–1047.

1. 1. In this study, the authors examined the effects of chronic (14 days) changes in thyroid function on a major neuromodulatory receptor system in the brain —the adenosinergic system. While previous investigators have examined the effects of alteration in thyroid function on adenosine receptors in peripheral tissues (adipocytes), this is the first study to examine such effects in brain.

2. 2. Three groups of male Sprague-Dawley rats were treated for 14 days with either a) oral PTU (0.00625%), iodine-free diet, and i.p. saline injections, b) i.p. saline injections, or c) i.p. triiodothyronine (25 μg/100 g) injections.

3. 3. These manipulations reliably resulted in the production of hypothyroidism (TSH 30.2 ± 8.6 ng/ml), euthyroidism (TSH 2.1 ± 0.9), and hyperthyroidism (TSH <0.4).

4. 4. Treatment had no significant effect on the B_max or K_d of [³H]DPCPX (A₁-antagonist) binding to homogenates from cerebral cortex, cerebellum or hippocampus; similarly, no effect on [³H]CGS-21680 (A₂-agonist) binding to striatal homogenates was noted.

5. 5. Similarly, quantitative autoradiographic studies failed to reveal consistent regional alterations unique to either hypo- or hyperthyroidism.

6. 6. Incubation of sections with GppNHp resulted in the expected reduction (≈ 40%) in agonist binding, but there was no differential effect seen for either the hypoor hyperthyroid tissues.

7. 7. These preliminary findings suggest that alterations in brain adenosine receptors or G-protein-receptor coupling are unlikely to be requisite correlates of abnormal thyroid hormone levels.

Striatal neurochemical changes in transgenic models of Huntington's disease

Transgenic mouse models of Huntington's disease (HD) were examined following the onset of overt behavioral symptoms. The HD transgenic mice demonstrated profound striatal losses in D1, D2, and D3 dopamine (DA) receptor proteins in comparison with their nonsymptomatic, age-matched littermate controls. In parallel, a robust increase in the striatal D5 DA receptor subtype occurred in the transgenic compared with the wild-type control mice. This receptor elevation was accompanied by heightened cyclic AMP levels, which may be induced by the adenylyl cyclase-linked D5 receptor. This is a unique result; normal striatal D5 protein levels are modest and not thought to contribute substantially to cyclic AMP-mediated DA signaling mechanisms. Simple compensatory up-regulation of D5 DA receptors in response to D1 receptor subtype loss does not explain our findings, because genetic inactivation of the D1 DA receptor does not alter levels of D5 DA receptor expression. Immunofluorescent detection of tyrosine hydroxylase showed that nigrostriatal DA containing terminals were reduced, further supporting that disturbances in DA signaling occurred in HD transgenic models. The substance P-containing striatal efferent pathway was more resistant to the HD mutation than met-enkephalin-producing striatal projection neurons in the transgenics, based on neuropeptide immunofluorescent staining. Analogous findings in multiple transgenic models suggest that these changes are due to the presence of the transgene and are not dependent on its composition, promotor elements, or mouse strain background. These findings suggest modifications in the striatal DA system and that its downstream signaling through cyclic AMP mechanisms is disrupted severely in HD following onset of motor symptoms.     

Association of serum levels of leptin,ghrelin, and adiponectin in schizophrenic patients and healthy controls

Abstract

Background. Leptin, ghrelin, and adiponectin play important roles in the regulation of body weight, food intake, and energy homeostasis, and have been suggested to be important biomarkers of metabolic syndrome. In this study, we tried to simultaneously investigate the serum levels of leptin, ghrelin, and adiponectin in schizophrenic patients and healthy controls. Methods. During a period of 2 years, we recruited 37 schizophrenic patients and 65 healthy controls. The levels of metabolic syndrome-related biomarkers including serum adiponectin, leptin, and ghrelin were measured with an enzyme-linked immunosorbent assay. Results. On applying analysis of covariance (ANCOVA) with age and body mass index adjustments, the leptin levels of schizophrenic patients (P = 0.038) were found to be higher than those of healthy controls. However, there were no significant differences in the serum levels of ghrelin or adiponectin between these two groups. Conclusion. These results showed that serum leptin levels might be more sensitive than ghrelin or adiponectin levels between schizophrenic patients and healthy controls. However, studies with a large sample size are needed to confirm these results.     

Fimbria-fornix lesion does not affect APP levels and amyloid deposition in the hippocampus of APP+PS1 double transgenic mice

The deposition of amyloid beta peptides (Abeta) and cholinergic dysfunction are two characteristic features of Alzheimer's disease. Several studies have suggested that a compromised cholinergic transmission can increase the amount of amyloid precursor protein (APP) in the denervated cortex (or hippocampus); however, whether this will increase Abeta production is unknown. To investigate the relation between cholinergic neurotransmission and APP metabolism, and the possible role of cholinergic dysfunction in the development of amyloid neuropathology, we lesioned the fimbria-fornix pathway in APP+PS1 double transgenic mice, at 5 and 7 months of age. Three months and 11 months postlesion, the mice were sacrificed for biochemical and histopathological analyses. The fimbria-fornix transection resulted in a substantial depletion of cholinergic markers in the hippocampus at both time points. Three months postlesion, hippocampal APP and Abeta levels were not significantly changed. At 11 months postlesion, the fimbria-fornix lesion did not result in an alteration in either the hippocampal Abeta levels or the extent of Abeta deposition, as assessed by amyloid plaque counts and image analysis of Abeta load in the 18-month-old APP+PS1 mice. Our findings indicate that APP metabolism in mice may be dissociated from cholinergic neurotransmission rather than related as previously suggested in other mammalian species.     

Nesfatin-1 suppresses energy intake, co-localises ghrelin in the brain and gut, and alters ghrelin, cholecystokinin and orexin mRNA expression in goldfish

Nesfatin‐1 is a novel anorectic peptide encoded in the precursor protein nucleobindin‐2 (NUCB2). We recently reported the presence and appetite suppressing effects of nesfatin‐1 in goldfish. Nesfatin‐1 has been co‐localised with ghrelin in the stomach of rats. Whether nesfatin‐1 influences other appetite regulatory peptides in goldfish remains unclear. The main objectives of the present study were to investigate whether nesfatin‐1 co‐localises ghrelin in goldfish, and to test whether exogenous nesfatin‐1 influences endogenous ghrelin, cholecystokinin (CCK) and orexin A (OXA). We found co‐localisation of nesfatin‐1‐like and ghrelin‐like immunoreactivity in the enteroendocrine cells of the goldfish anterior intestine (J‐loop). Furthermore, co‐localisation of ghrelin and nesfatin‐1 was also observed in the posterior nucleus lateralis tuberis of the goldfish hypothalamus, a brain region implicated in the regulation of food intake. These findings suggest a functional relationship between ghrelin and nesfatin‐1 in goldfish. In support of this, i.c.v. administration of goldfish (gf) nesfatin‐1 [25 ng/g body weight (BW)], suppressed food intake and the expression of mRNAs encoding preproghrelin, ghrelin receptor (GHS‐R 1a‐1), CCK and NUCB2 in the forebrain of fed fish, as well as ghrelin and NUCB2 mRNA in the hypothalamus of unfed fish, both at 1 h post‐injection. Nesfatin‐1 stimulated hypothalamic CCK mRNA expression at 30 min post‐injection in fed fish, and inhibited OXA mRNA in the unfed fish hypothalamus 1 h post‐injection. Similarly, i.c.v. injections of gfghrelin (1 ng/g BW), although stimulating food intake, suppressed NUCB2 and preproghrelin mRNAs, but not ghrelin receptor mRNA expression in the forebrain. It is also evident that exogenous ghrelin and nesfatin‐1 mRNAs encoding these peptides. Our novel results indicate interactions between nesfatin‐1 and ghrelin, CCK and orexin, and show that nesfatin‐1 acts on other appetite regulatory peptides in a time‐ and feeding status‐dependent, as well as tissue‐specific, manner in goldfish.     

Physiological and induced neuronal death are not affected in NSE-bax transgenic mice

Bax, a family member of the survival protein Bcl-2, is expressed in the nervous system during development and throughout adulthood. Bax deficiency has been demonstrated to prevent developmental and trophic factor deprivation-induced neuronal death. To further clarify the role of Bax in naturally occurring neuronal death and in neuronal death following apoptotic stimuli, we generated several lines of transgenic mice expressing the human Bax protein specifically in neurons, under the control of the neuron-specific enolase promoter. Transgene expression was first detected around E10.5 and E12.5, depending on the transgenic line. The total number of ganglion cells in the retina and of pyramidal cells in the hippocampus, both expressing the transgene, was similar in control and transgenic mice. In addition, in our model system, Bax overexpression did not appear to influence the in vitro survival of sensory neurons isolated from dorsal root ganglia after nerve grwoth factor (NFG) deprivation or the apoptotic death of motor neurons following axotomy. J. Neurosci. Res. 52:247–259, 1998. © 1998 Wiley-Liss, Inc.     

Acute glucocorticoid pretreatment suppresses stress-induced hypothalamic-pituitary-adrenal axis hormone secretion and expression of corticotropin-releasing hormone hnRNA but does not affect c-fos mRNA or fos protein expression in the paraventricular nucleus of the hypothalamus

Corticosterone regulates both basal and stress-induced hypothalamic-pituitary-adrenal (HPA) axis activity in a negative-feedback fashion. However, the cellular and molecular mechanisms of this negative feedback have yet to be explicitly characterized. By comparing stress-induced c-fos and corticotropin-releasing hormone (CRH) expression in the paraventricular nucleus (PVN), we may be able to determine whether acute glucocorticoid treatment affects the net neural excitatory input to the PVN (represented primarily by c-fos mRNA expression) or directly affects the ability of cells in the PVN to respond to that input (represented primarily by CRH hnRNA expression). In the following studies, we observed the effect of acute glucocorticoid (RU28362) treatment on subsequent HPA axis reactivity by measuring stress-induced plasma hormone concentration [corticosterone and adrenocorticotropic hormone (ACTH)] and gene expression (c-fos and CRH) in the PVN. First, we examined the dose-response relationship between systemically administered RU28362 (1-150 microg/kg, i.p) and suppression of the stress-induced corticosterone response. We then confirmed central nervous system access of the maximally suppressive dose of RU28362 (150 microg/kg) by an ex vivo radioligand binding assay. RU28362 selectively occupied the majority of glucocorticoid receptors in the hippocampus and hypothalamus while having no effect on mineralocorticoid receptors. In separate studies, RU28362 (150 microg/kg) and corticosterone (5 mg/kg) were injected i.p. 1 h before restraint stress. Compared to vehicle-treated controls, rats treated with RU28362 and corticosterone had substantially blunted stress-induced corticosterone and ACTH production, respectively. Furthermore, treatment with RU28362 significantly blunted stress-induced CRH hnRNA expression in the PVN. By contrast, neither RU28362 nor corticosterone treatment had an effect on stress-induced neuronal activation as measured by c-fos mRNA and its protein product in the PVN. This dissociation between c-fos and CRH gene expression suggests that glucocorticoid suppression of HPA activity within this time-frame is not a result of decreased excitatory neural input to the PVN, but instead depends on some direct effect of RU28362 on cells intrinsic to the HPA axis.     

GFAP promoter driven transgenic expression of PDGFB in the mouse brain leads to glioblastoma in a Trp53 null background

Glioblastomas are the most common and malignant astrocytic brain tumors in human adults. The tumor suppressor gene TP53 is commonly mutated and/or lost in astrocytic brain tumors and the TP53 alterations are often found in combination with excessive growth factor signaling via PDGF/PDGFRα. Here, we have generated transgenic mice over‐expressing human PDGFB in brain, under control of the human GFAP promoter. These mice showed no phenotype, but on a Trp53 null background a majority of them developed brain tumors. This occurred at 2–6 months of age and tumors displayed human glioblastoma‐like features with integrated development of Pdgfrα⁺ tumor cells and Pdgfrβ⁺/Nestin⁺ vasculature. The transgene was expressed in subependymal astrocytic cells, in glia limitans, and in astrocytes throughout the brain substance, and subsequently, microscopic tumor lesions were initiated equally in all these areas. With tumor size, there was an increase in Nestin positivity and variability in lineage markers. These results indicate an unexpected plasticity of all astrocytic cells in the adult brain, not only of SVZ cells. The results also indicate a contribution of widely distributed Pdgfrα⁺ precursor cells in the tumorigenic process. © 2008 Wiley‐Liss, Inc.     

Signaling of ghrelin and its functional receptor,the growth hormone secretagogue receptor,promote tumor growth in glioblastomas

Ghrelin is a 28‐amino‐acid peptide that is the endogenous ligand for the pituitary growth hormone secretagogue receptor (GHS‐R). Ghrelin is mainly produced from the stomach, but it is also expressed by various other tissues, including the CNS under normal conditions. Physiologically, ghrelin regulates appetite, gut motility, and GH release from the anterior pituitary, as well as cardiovascular and immune systems. Recent studies also indicate that ghrelin and GHS‐R may play an important autocrine/paracrine role in neoplastic conditions. In order to clarify the role of ghrelin/GHS‐R in gliomas, the present study assessed the expression of ghrelin and its functional receptor, GHS‐R1a, in 39 glioblastomas (GBs), 13 anaplastic astrocytomas (AAs) and 11 diffuse astrocytomas (DAs) using immunohistochemical analyses. Immunohistochemical staining was evaluated as follows: no staining; 1+, 0–10% positive cells; 2+, 10–50% positive cells; 3+, >50% positive cells. Ghrelin expression was detected in 52 of 63 cases of which 38, 13 and one were scored as 3+, 2+ and 1+, respectively. GHS‐R1a expression was detected in 45 of 63 cases of which 29, 15 and one were scored as 3+, 2+ and 1+, respectively. Ghrelin immunoreactivity was observed in 38 of 39 GBs, 12 of 13 AAs and two of 11 DAs. GHS‐R1a immunoreactivity was observed in 39 of 39 GBs, five of 13 AAs, and one of 11 DAs. AAs and GBs showed moderate or strong immunostaining of ghrelin/GHS‐R1a in the tumor cells and in proliferating microvessels. Patients were classified into lower to moderate‐score, and high‐score ghrelin/GHS‐R categories according to the principal component and cluster analyses. Multivariate analysis of overall survival indicated that there was a significant difference (P = 0.0001) in the survival rate between these two groups. The combined results indicated that expression of the ghrelin/GHS‐R1a axis increases the growth of AAs and GBs through an autocrine/paracrine mechanism.     

Neurone-specific expression and regulation of the pufferfish isotocin and vasotocin genes in transgenic mice

We used comparative genetics to investigate the location, structure and evolution of the oxytocin and vasopressin gene regulatory regions. The pufferfish, Fugu rubripes, is an attractive vertebrate model for comparison because of its maximal evolutionary distance from mammals and short intergenic regions. To determine whether regulatory DNA is conserved between oxytocin and vasopressin, and their Fugu homologs, isotocin and vasotocin, we generated transgenic mice bearing overlapping Fugu cosmids that contained the isotocin and/or vasotocin genes as well as short isotocin (5 kb) and vasotocin (9 kb) constructs. Our study shows that the Fugu isotocin and vasotocin genes express specifically in the mouse oxytocinergic and vasopressinergic neurones, respectively, and that the cis-regulatory elements which mediate neurone-specific expression are located within the short transgene constructs tested. Thus, the neurone-specific expression of the oxytocin and vasopressin gene families, and the mechanisms mediating the cell-specificity, evolved before the divergence of the fish and mammalian lineages. Salt-loading of transgenic mice induced an increase in abundance of isotocin, but not vasotocin mRNA in the cognate neurones. It appears that either the vasotocin gene does not respond to osmotic perturbations or the vasotocin transgene construct tested lacks osmotic response elements. Comparisons of homologous flanking sequences of the Fugu and mouse genes identified several short matching sequences, which are candidate regulatory elements.     

Adrenocorticotrophic hormone and melanocyte stimulating hormone do not affect performance in hippocampus-lesioned or control pigeons

Pigeons, given bilateral lesions of the hippocampus, as well as unoperated control subjects, were trained on an operant task in which they had to peck a left hand key if both members of a successively presented stimulus pair were the same, and a right hand key if they were different. During extensive training, the 'same' pairs (green-green or red-red) were presented at twice the frequency of the 'different' pairs (green-red or red-green), thus biasing responding to the left hand key. The effects of intramuscular (i.m.) injections (0, 10 and 100 micrograms/kg) of an active fragment of adrenocorticotrophic hormone (ACTH4-10) and alpha-melanocyte stimulating hormone (MSH) were then studied. On any given test (peptide or saline) day, all stimulus pairs were presented with equal frequency. Although the control birds' performance was consistently better than the lesioned birds, neither ACTH4-10 nor MSH affected performance under any conditions. It is concluded that peripheral injection of these peptides has little, if any, effect on cognitive behaviour in birds.     

Anatomically specific changes in the expression of somatostatin, growth hormone-releasing hormone and growth hormone receptor mRNA in diabetic rats

Growth hormone (GH) secretion is altered in poorly controlled diabetic animals. However, modifications in the hypothalamic neuropeptides that control GH secretion, somatostatin and GH-releasing hormone (GHRH), as well as changes in the sensitivity of the hypothalamus and pituitary to the feedback effects of GH, are less clear. We have used RNase protection assays and in-situ hybridization to address whether the mRNA expression of GH, somatostatin and GHRH, as well as of the GH receptor (GHR) in the hypothalamus and anterior pituitary, are altered in streptozotocin-induced diabetic rats. After induction of diabetes, rats were treated with insulin twice daily for 3 weeks to obtain either poorly controlled (mean plasma glucose >300 mg/dl) or well-controlled diabetic rats. Although no significant change in pituitary GH mRNA expression was found, the hypothalamic expression of GHRH and somatostatin mRNA was reduced in poorly-controlled diabetic rats and returned to control values with normalisation of plasma glucose concentrations (P<0.0001 and P<0.002, respectively). Somatostatin mRNA expression was reduced only in the central portion of the periventricular nucleus, with no change being seen in the other areas of the periventricular nucleus or in the arcuate, suprachiasmatic or paraventricular nuclei. A significant decline in GHRH mRNA expression was observed in both the arcuate nucleus and ventromedial hypothalamus. Anterior pituitary GHR mRNA expression was significantly reduced in both well and poorly-controlled diabetic rats, while there was no change in the hypothalamus. To examine whether the evolution time of the diabetes influences these parameters, in a subsequent experiment, diabetic rats received no insulin for 2 months. A significant decline in GHRH and somatostatin mRNA expression was also observed in these rats. In addition, pituitary GH mRNA expression declined significantly in long-term diabetic rats. These results demonstrate that: (1) the expression of both GHRH and somatostatin declines specifically in anatomical areas involved in anterior pituitary hormone control; (2) GHR mRNA expression is decreased in the pituitary of diabetic rats, but not in the hypothalamus, and does not return to control values with normalisation of mean blood glucose concentrations; and (3) the evolution time of the diabetes is important for detecting some changes, including the decrease in pituitary GH mRNA expression.