Abstract: Growth hormone (GH)-deficiency is usually associated with elevated adiposity, hyperleptinemia, and increased fracture risk. Since leptin is thought to enhance cortical bone formation, we have investigated the contribution of elevated adiposity and hyperleptinemia on femoral strength in rodent models of GH deficiency. Quantification of the transpubertal development of femoral strength in the moderately GH-deficient/hyperleptinemic Tgr rat and the profoundly GH-deficient/hypoleptinemic dw/dw rat revealed that the mechanical properties of cortical bone in these two models were similarly compromised, a 25-30% reduction in failure load being entirely due to impairment of geometric variables. In contrast, murine models of partial (GH antagonist transgenic) and complete (GH receptor-null) loss of GH signaling and elevated adiposity showed an impairment of femoral cortical strength proportionate to the reduction of GH signaling. To determine whether impaired femoral strength is exacerbated by obesity/hyperleptinemia, femoral strength was assessed in dw/dw rats following two developmental manipulations that elevate abdominal adiposity and circulating leptin, neonatal monosodium glutamate (MSG) treatment, and maintenance on an elevated fat diet. The additional impairment of femoral strength following MSG treatment is likely to have resulted from a reduction in residual activity of the hypothalamo-pituitary-GH-IGF-I axis, but consumption of elevated dietary fat, which did not reduce circulating IGF-I, failed to exacerbate the compromised femoral strength in dw/dw rats. Taken together, our data indicate that the obesity and hyperleptinemia usually associated with GH deficiency do not exert a significant influence over the strength of cortical bone.

Abstract: Circulating ghrelin elevates abdominal adiposity by a mechanism independent of its central orexigenic activity. In this study we tested the hypothesis that peripheral ghrelin induces a depot-specific increase in white adipose tissue (WAT) mass in vivo by GH secretagogue receptor (GHS-R(1a))-mediated lipolysis. Chronic iv infusion of acylated ghrelin increased retroperitoneal and inguinal WAT volume in rats without elevating superficial sc fat, food intake, or circulating lipids and glucose. Increased retroperitoneal WAT mass resulted from adipocyte enlargement probably due to reduced lipid export (ATP-binding cassette transporter G1 mRNA expression and circulating free fatty acids were halved by ghrelin infusion). In contrast, ghrelin treatment did not up-regulate biomarkers of adipogenesis (peroxisome proliferator-activated receptor-gamma2 or CCAAT/enhancer binding protein-alpha) or substrate uptake (glucose transporter 4, lipoprotein lipase, or CD36) and although ghrelin elevated sterol-regulatory element-binding protein 1c expression, WAT-specific mediators of lipogenesis (liver X receptor-alpha and fatty acid synthase) were unchanged. Adiposity was unaffected by infusion of unacylated ghrelin, and the effects of acylated ghrelin were abolished by transcriptional blockade of GHS-R(1a), but GHS-R(1a) mRNA expression was similar in responsive and unresponsive WAT. Microarray analysis suggested that depot-specific sensitivity to ghrelin may arise from differential fine tuning of signal transduction and/or lipid-handling mechanisms. Acylated ghrelin also induced hepatic steatosis, increasing lipid droplet number and triacylglycerol content by a GHS-R(1a)-dependent mechanism. Our data imply that, during periods of energy insufficiency, exposure to acylated ghrelin may limit energy utilization in specific WAT depots by GHS-R(1a)-dependent lipid retention.

Abstract: With one quarter of the population of the Western world now considered obese, it is essential that we understand the factors giving rise to elevated fat deposition. This review summarizes the cellular and molecular mechanisms governing the volume of white adipose tissue (WAT), and outlines the physiological signals that regulate these processes. Particular attention is given to the role of the gastric hormone, ghrelin, describing its actions in general and presenting detailed evidence of its role in regulating adipocyte biology. Combining this evidence with an analysis of the factors governing ghrelin secretion, leads to the hypothesis that during periods of food deprivation ghrelin acts as an energy deficit signal, defending the fat stored in responsive WAT against the forces of utilization. This scenario has clear implications for programmes of sustainable weight loss.

Abstract: Liver X receptors (LXRs), activated by oxysterols, play an important role in the regulation of lipid and glucose metabolism, which is also markedly dependent on thyroid hormone and growth hormone (GH) status. Here, we investigated how a 1-week exposure to the synthetic LXR agonist T0901317 affected GH secretion and thyroid hormone status in male rats. While the pulse frequency of GH secretion was marginally affected there was a highly significant decrease in the triiodo-L-thyronine/thyroxine (T3/T4) ratio in plasma. This effect was associated with decreased expression of deiodinase 1 (DIO1) and 2 (DIO2) mRNA in the liver and thyroid gland, respectively. Expression of sterol regulatory element binding protein-1c (SREBP-1c), the hallmark of stimulated lipogenesis, was markedly increased in both thyroid and pituitary implying that protracted pharmacological LXR activation may promote lipid accumulation in these endocrine tissues. These findings suggest that attention must be given to pituitary hormone dependent axes when developing therapeutic strategies based on agonism of the LXRs, e.g. for treatment of atherosclerosis.

Abstract: 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.

Abstract: Arylalkylamine N-acetyltransferase (Aanat) is the penultimate enzyme in the serotonin-N-acetylserotonin-melatonin pathway. It is nearly exclusively expressed in the pineal gland and the retina. A marked rhythm of Aanat gene expression in the rat pineal is mediated by cyclic AMP response elements located in the promoter and first intron. Intron 1 also contains E-box elements, which mediate circadian gene expression in other cells. Here we examined whether these elements contribute to rhythmic Aanat expression in the pineal gland. This was done using transgenic rats carrying Aanat transgenes with mutant E-box elements. Circadian expression of Aanat transgenes was not altered by these mutations. However, these mutations enhanced ectopic expression establishing that the intronic Aanat E-box elements contribute to the gene's pineal specific expression. A similar role of the Aanat E-box has been reported in zebrafish, indicating that Aanat E-box mediated silencing is a conserved feature of vertebrate biology.

Abstract: This study describes the previously uncharacterized ontogeny and regulation of truncal adipose reserves in the profoundly GH-deficient dwarf (dw/dw) rat. We show that, despite normal proportionate food intake, dw/dw rats develop abdominal leanness and hypoleptinemia (circulating leptin halved in dw/dw males, P < 0.05) during puberty. This contrasts with the hyperleptinemia seen in moderately GH-deficient Tgr rats (circulating leptin doubled at 6 wk of age, P < 0.05) and in GH receptor-binding protein (GHR/BP)-null mice (circulating leptin doubled; P < 0.05). This lean/hypoleptinemic phenotype was not completely normalized by GH treatment, but dw/dw rats developed abdominal obesity in response to neonatal MSG treatment or maintenance on a high-fat diet. Unlike Tgr rats, dw/dw rats did not become obese with age; plasma leptin levels and fat pad weights became similar to those in wild-type rats. In contrast with truncal leanness, tibial marrow adiposity was normal in male and doubled in female dwarves (P < 0.01), this increase being attributable to increased adipocyte number (P < 0.01). Neonatal MSG treatment and high-fat feeding elevated marrow adiposity in dw/dw rats by inducing adipocyte enlargement (P < 0.05). These results demonstrate that, despite lipolytic influence of GH, severe GH deficiency in dw/dw rats is accompanied by a paradoxical leanness. This lean/hypoleptinemic phenotype is not solely attributable to reduced GH signaling and does not appear to result from a reduction in nutrient intake or the ability of dw/dw adipocytes to accumulate lipid. Disruption of preadipocyte differentiation or adipocyte proliferation in the dw/dw rat may lead to the development of this unusually lean/hypoleptinemic phenotype.

Abstract:

Abstract: The inducible transcription factor Egr-1 has been extensively studied in the adult brain but potential roles during development are largely unexplored. Here we describe the analysis of a new transgenic rat model (egr-1 promoter driving a destabilized GFP molecule) that has provided novel information about the postnatal roles of Egr-1. We show that Egr-1 is more widely expressed in the neonatal brain than was previously appreciated, and is not restricted to neurons; it is expressed in glial cells in the postnatal neocortex and hippocampus. This pattern of expression has been revealed due to cellular filling by GFP, permitting co-localization with glial markers. The transgene/Egr-1 is also expressed in a novel population of cells associated with Cajal-Retzius-like neurons within the marginal zone of the postnatal neocortex. Both of these cellular populations are transient, being limited to the neonatal period, before Egr-1 expression becomes established in an adult-like pattern within neocortical neurons, CA1 hippocampus, and striatum. Another transient population of transgene/Egr-1 cells in the bed nucleus of the stria terminalis is maintained until pre-adolescence. The transient phenotype of these cells involves a low relative expression of the neuronal marker NeuN, perhaps indicating a failure to achieve full neuronal differentiation. Egr-1 is therefore present in a diverse range of cell-types during postnatal development. Transgenic expression of a destabilized fluorescent marker has permitted identification of these novel cell populations and will facilitate further analysis of the transcriptional mechanisms that underlie the specific functions and fate of these cells during postnatal brain development.

Abstract: Sparc null mutants have been generated independently via targeted mutations in exons 4 and 6. Previous studies have identified low-turnover osteopenia in the 129Sv/C57BL/6 exon 4 knockout. Since both Sparc null mutations result in complete absence of Sparc protein, similar phenotypic outcomes are likely. However, genetic background (strain) and/or linkage disequilibrium effects can influence phenotype. Different inactivating mutations should be tested in various mouse strains; similar phenotypic outcomes can then confidently be assigned to the mutated gene. We have evaluated the bone phenotype in the 129Sv/EvSparc(tm1cam) exon 6 knockout at 4 and 9 mo, using physical measurement, mechanical strength tests, and DXA scanning. We have also quantified bone marrow adiposity and circulating leptin levels to assess adipose tissue metabolism. 129Sv/EvSparc(tm1cam) null mice show decreased bone mineral density and bone mineral content and increased mechanical fragility of bone, in line with previous studies. Differences were also noted. Increased body weight and levels of bone marrow adiposity but decreased circulating leptin concentrations were identified at 4, but not 9 mo, and 129Sv/EvSparc(tm1cam) null mice also had shorter femurs. Molecular phenotyping was carried out using mouse HGMP NIA microarrays with cortical femur samples at various ages, using semiquantitative RT-PCR validation. We identified 429 genes highly expressed in normal bone. Six genes (Sparc, Zfp162, Bysl, E2F4, two ESTs) are differentially regulated in 129Sv/EvSparc(tm1cam) cortical femur vs. 129Sv/Ev controls. We confirm low-turnover osteopenia as a feature of the Sparc null phenotype, identifying the usefulness of this mouse as a model for human osteoporosis.

Abstract: Ghrelin, the endogenous ligand of GH secretagogue receptor type 1a, has emerged as pleiotropic modulator of diverse biological functions, including energy homeostasis and, recently, reproduction. Although inhibitory actions of ghrelin on LH secretion and puberty onset have been reported previously, the receptor mechanisms mediating these actions, and the potential gonadotropic effects of the unacylated isoform of ghrelin (UAG), remain unclear. In this work, the effects of single and repeated administration of ghrelin or UAG on LH secretion were compared in pubertal and adult male rats. In addition, the effects of ghrelin were assessed in models of transient or persistent hypergonadotropism. Daily injection of ghrelin or UAG throughout puberty similarly decreased LH levels and partially delayed balanopreputial separation. Likewise, chronic infusion of ghrelin or UAG to adult males resulted in significant decreases in circulating LH and FSH concentrations. Moreover, acute injection of ghrelin induced a transient reduction in LH levels in freely moving males, an effect that was fully mimicked by administration of UAG. Yet in contrast to ghrelin, UAG failed to modify GH secretion. Finally, injection of ghrelin moderately, but significantly, reduced the duration of LH secretory responses to the potent gonadotropin secretagogue kisspeptin-10, whereas ghrelin infusion in a model of chronic elevation of serum gonadotropin levels (the transgenic growth retarded male rat) evoked a significant reduction of LH concentrations. Altogether our present results further substantiate the inhibitory effect of ghrelin on basal and stimulated LH secretion in a wide array of experimental conditions. Moreover, our data are the first to demonstrate the ability of UAG, originally considered an inert form of the molecule, to mimic the actions of acylated ghrelin on LH release. These observations reinforce the contention that ghrelin, as putative signal for energy insufficiency, may operate as negative modifier of male puberty and LH secretion, an effect that might be, at least partially, conducted through a GH secretagogue receptor type 1a-independent mechanism.

Abstract: Growth hormone (GH) is known to regulate peripheral components of the hypothalamo-pituitary gonadal (HPG) axis, but it remains unclear whether GH exerts a significant influence on the activity of the hypothalamo-pituitary components of the HPG axis. In this study, we investigated the development of HPG axis function in the male transgenic growth retarded (Tgr) rat, a model of moderate systemic GH deficiency caused by hypothalamic expression of human (h)GH. Impaired postnatal somatotroph expansion and moderate GH deficiency in male Tgr rats were accompanied by a two- to three-fold increase in pituitary gonadotrophin content, but without a significant change in the pituitary gonadotroph population. A three- to nine-fold elevation in basal circulating luteinising hormone concentration was seen in postpubertal Tgr rats, with a smaller increase in follicle-stimulating hormone. Despite this hypergonadotrophism, there was no corresponding increase in steroidogenic (circulating testosterone and seminal vesicle weights) or gametogenic (spermatozoa counts in seminiferous tubules) activity in the postpubertal Tgr testis. Following puberty, the plasma leptin concentration also became progressively elevated in Tgr males. Circulating gonadotrophin and leptin levels were normalised in Tgr rats by peripheral physiological replacement of rat GH, but plasma testosterone concentration was unaffected. These results confirm that hGH exerts a positive influence on the central control of gonadotrophin secretion in the Tgr rat, but the absence of a corresponding elevation in the steroidogenic or gametogenic function of the Tgr testis implies that the peripheral GH/insulin-like growth factor I axis may also exert a permissive influence on testicular function. The relative contribution of somatogenic and lactogenic mechanisms and the potential influence of elevated leptin and decreased sensitivity to androgen feedback to the development of postpubertal hypergonadotrophism in Tgr males remain to be determined.

Abstract: Profound somatotroph hypoplasia in the dwarf (dw/dw) rat is accompanied by an estrogen-dependent induction of prolactin secretion by the GH secretagogue, GHRP-6. Using electron microscopy, we demonstrated that the reduction in the somatotroph population in the dw/dw pituitary is accompanied by the presence of a morphologically distinct lactotroph subpopulation. In these cells, which did not coexpress GH, the size, shape, and number of the secretory granules were between those of the type I and type II lactotrophs. We therefore called these cells intermediate lactotrophs. The intermediate lactotrophs accounted for up to 30% of the total prolactin-positive cell population in dw/dw males and up to 12% in females. Using tannic acid to quantify the fusion of secretory granules, we have shown that the intermediate lactotrophs are unresponsive to either GH-releasing factor (GRF) or TRH but exhibit a sexually dimorphic secretory response to acute ghrelin treatment, granular fusions being 4-fold higher in females. No cell matching the morphology of the novel lactotroph subpopulation was observed in the pituitary of the GRF-insensitive lit/lit mouse. However, ablation of GRF neurons with neonatal monosodium glutamate treatment had no effect on the population of intermediate lactotrophs in the dw/dw rat. Thus, the presence of the intermediate lactotrophs in the dw/dw pituitary appears to be independent of the function of the GRF neurons.

Abstract: Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.

Abstract: It is well established that photic cues are used by the suprachiasmatic nucleus (SCN) to entrain circadian rhythms to the light/dark cycle, but the role of photic stimuli in the regulation of ultradian neuroendocrine rhythms is ill defined. In relation to the rhythms of GH secretion, recent studies have shown that nocturnal photic stimulation induces gene expression not only in the SCN but also in periventricular (PeN) somatostatin (SRIF) neurons. We have, therefore, investigated the effect of nocturnal photic stimulation on spontaneous and induced GH secretion in conscious rats. Nocturnal photic stimulation (lights on at 2400 h for 1 h) suppressed spontaneous GH secretion in male and female rats and reduced the GH response to SRIF withdrawal and iv injection of GH-releasing factor. A similar trough in GH secretion was also observed during the first hour of the normal light phase (0600 h). Using immunohistochemical analysis, we have also shown that expression of the transcription factor, Egr-1, is induced at the commencement of the light phase in the SCN, PeN, and medial preoptic nucleus. This effect is abolished by maintaining rats in the dark during this period. These data, together with our previous demonstration that 50% of SRIF-positive neurons in the PeN coexpress Egr-1 after photic stimulation, suggest that activation of SRIF neurons in the PeN may entrain the episodes of GH secretion to the dark/light interface. However, the absence of synchrony in GH pulses between animals by the second half of the light period suggests that this entrainment is transient.

Abstract: Ghrelin promotes fat accumulation, despite potent stimulation of the lipolytic hormone, GH. The function of the major circulating isoform of ghrelin, des-octanoyl ghrelin, is unclear, because it does not activate the GH secretagogue receptor (GHS-R1a) and lacks the endocrine activities of ghrelin. We have now addressed these issues by infusing ghrelin, des-octanoyl ghrelin, or synthetic GHS-R1a agonists into three rat models with moderate, severe, or total GH deficiency. We show that in the context of significant GH secretion, the adipogenic effect of systemic ghrelin infusion is pattern dependent. However, this adipogenic action is not mediated by the pituitary hormones. Using a novel unilateral local infusion strategy, we demonstrate that ghrelin promotes bone marrow adipogenesis in vivo by a direct peripheral action. Surprisingly, this effect was also observed with des-octanoyl ghrelin, whereas a potent synthetic GHS-R1a agonist was ineffective. Thus, these adipogenic effects are mediated by a receptor other than GHS-R1a. This is the first in vivo demonstration of a direct adipogenic effect of des-octanoyl ghrelin, a major circulating form of ghrelin that lacks GH-releasing activity. We suggest that the ratio of ghrelin and des-octanoyl ghrelin production could help regulate the balance between adipogenesis and lipolysis in response to nutritional status.

Abstract: The extent to which childhood GHD affects adult fracture risk is unclear. We measured femoral strength in adult transgenic growth-retarded rats as a model of GHD. Long-term, moderate GHD was accompanied by endocrine and morphometric changes consistent with a significant reduction in femoral strength. INTRODUCTION: Childhood growth hormone deficiency (GHD) is associated with osteopenia, but little is known about its effects on subsequent adult bone strength and fracture risk. MATERIALS AND METHODS: We have therefore measured femoral strength (failure load measured by three-point bending) in a new model of moderate GHD, the transgenic growth-retarded (Tgr) rat at 15, 22-23, and 52 weeks of age, and have quantified potential morphological and endocrine determinants of bone strength. RESULTS: Skeletal growth retardation in Tgr rats was accompanied by a sustained reduction in the anterior-posterior diameter of the femoral cortex, whereas mid-diaphyseal cortical wall thicknesses were largely unaltered. Total femoral strength was significantly impaired in Tgr rats (p < 0.01), and this impairment was more pronounced in males than females. Compromised bone strength in Tgr rats could not be accounted for by the reduction in mechanical load (body weight) and was not caused by impairment of the material properties of the calcified tissue (ultimate tensile stress), despite marked reductions in femoral mineral density (areal bone mineral density; p < 0.001). Microcomputerized tomographical analysis revealed significant modification of the architecture of trabecular bone in Tgr rats, with reductions in the number and thickness of trabeculae (p < 0.05) and in the degree of anisotropy (p < 0.01). The marked reduction in plasma insulin-like growth factor-1 in Tgr rats was accompanied by the development of high circulating leptin levels (p < 0.01). CONCLUSION: These results show that the changes in endocrinology and bone morphology associated with long-term moderate GHD in Tgr rats are accompanied by changes consistent with a significant reduction in the threshold for femoral fracture.

Abstract: The peptide hormone ghrelin binds to the GH secretagogue receptor (GHS-R), stimulates GH secretion, and promotes adipogenesis. However, continuous GHS infusion does not stimulate skeletal growth and is associated with desensitization to further GH secretagogue treatment. In this study, 7-d intermittent (i.e. every 3 h) infusion of ghrelin, or the GH secretagogue, GH-releasing peptide-6, in the moderately GH- deficient transgenic growth-retarded rat, augmented GH secretion, leading to a sustained acceleration in skeletal growth. In contrast, continuous infusion of ghrelin, or GH-releasing peptide-6, suppressed the amplitude of spontaneous GH secretory episodes and produced only a transient increase in body weight gain. The reduction in GH secretion seen with continuous GHS-R activation was not associated with a desensitization of the pituitary to GH-releasing factor or to down-regulation of hypothalamic GHS-R mRNA expression. Continuous ghrelin treatment elicited an increase in somatostatin mRNA expression in the periventricular nuclei. Thus, exposure to continuously elevated circulating ghrelin may be responsible for the suppression of GH secretion reported in rats after prolonged starvation.

Abstract: Regulated expression of Egr-1 (Zif268/NGFIA) in a variety of brain networks suggests a diversity of roles in neuronal plasticity. Here, we aimed to determine whether an egr-1 transgene would mediate transcriptional responses to different pharmacological and physiological stimuli in the brain of transgenic rats. Constitutive transgene expression was observed in the cortex, CA1 hippocampal area and pituitary, recapitulating expression of egr-1. Transgene induction was stimulus-specific. Metrazole induced widespread expression in the dentate gyrus, CA2 and CA3 areas, amygdala, and ventromedial nucleus. In contrast, induction following a light stimulus was restricted to the hypothalamic suprachiasmatic and periventricular nuclei. Our studies have provided novel insights into the differential regulation of egr-1, and facilitated approaches to the genetic manipulation of Egr-1-regulated neuronal systems.

Abstract: Fos-related antigen 2 (Fra-2) is a member of the Fos family of immediate-early genes, most of which are rapidly induced by second messengers. All members of this family act by binding to AP-1 sites as heterodimeric complexes with other proteins. However, each appears to have a distinct role. The role and biology of Fra-2 are less well understood than those of its relatives c-Fos, Fra-1, and FosB; moreover, Fra-2 target genes remain largely unknown, as does the basis of its selective effects on transcriptional activity. To pursue these issues, we created a transgenic rat line (NATDNF2) in which a dominant negative fra-2 (DNF2) gene is strongly expressed in the pineal gland; tissue selectivity was achieved by putting the DNF2 gene under the control of the rat arylalkylamine N-acetyltransferase (AANAT) regulatory region, which targets gene expression to a very restricted set of tissues (pineal gland >> retina). Expression of AANAT is normally turned on after the onset of darkness in the rat; as a result, pineal DNF2 expression occurs only at night. This was associated with marked suppression of the nocturnal increase in fra-2 mRNA and protein levels, indicating that DNF2 expression inhibits downstream effects of Fra-2, including the maintenance of high levels of fra-2 gene expression. Analysis of 1,190 genes in the NATDNF2 pineal gland, including the AANAT gene, identified two whose expression is strongly linked to fra-2 expression: the genes encoding type II iodothyronine deiodinase and nectadrin (CD24).

Abstract: The transgenic growth retarded (Tgr) rat is the first genetic model of growth hormone (GH) deficiency whose growth can be accelerated with exogenous GH secretagogues (GHSs). In this study, we have demonstrated that GHS-receptor (GHS-R) mRNA expression in the arcuate nucleus of Tgr rats was not significantly different to that in wild-type littermates. We have confirmed that GHS-induced elevation in body weight gain was accompanied by acceleration of skeletal growth, and that the effects of the GHS, GHRP-6, were both dose- and pattern-dependent. The growth response with continuous infusion of GHRP-6 was transient, accompanied by suppression of GH and corticosterone responses to bolus injection of GHRP-6. This desensitization occurred without downregulation of arcuate GHS-R mRNA expression, but was accompanied by elevated periventricular somatostatin mRNA expression. In contrast, pulsatile (3-hourly) infusion of GHRP-6 produced sustained growth and GH responses, which were accompanied by suppression of corticosterone responses and elevated arcuate GH-releasing factor (GRF) mRNA expression. Skeletal growth was further accelerated by coinfusion of GRF, but significant depletion of pituitary GH stores suggested that this growth rate may not be sustainable. These experiments confirm the importance of the Tgr rat for investigating the growth promoting potential of the GHSs in the context of GH-deficient dwarfism, and suggest that elevated somatostatin expression may mediate the suppression of the GRF-GH and hypothalamo-pituitary-adrenal axes following continuous GHRP-6 treatment.

Abstract: As mammalian genome projects move towards completion, the attention of molecular neuroscientists is currently moving away from gene identification towards both cell-specific gene expression patterns (neuronal transcriptions) and protein expression/interactions (neuronal proteomics). In the long term, attention will increasingly be directed towards experimental interventions which are able to question neuronal function in a sophisticated manner that is cognisant of both transcriptomic and proteomic organization. Central to this effort will be the application of a new generation of transgenic approaches which are now evolving towards an appropriate level of molecular, temporal and spatial resolution. In this review, we summarize recent developments in transgenesis, and show how they have been applied in the principal model species for neuroscience, namely rats and mice. Current concepts of transgene design are also considered together with an overview of new genetically-encoded tools including both cellular indicators such as fluorescent activity reporters, and cellular regulators such as dominant negative signalling factors. Application of these tools in a whole animal context can be used to question both basic concepts of brain function, and also current concepts of underlying dysfuction in neurological diseases.

Abstract: The bacterial gene chloramphenicol acetyltransferase (CAT) is a widely used reporter in both in-vitro and in-vivo studies of genetic regulation. We have recently generated novel rat transgenic lines carrying an arylalkylamine N-acetyltransferase (AA-NAT) promoter-reporter construct in which CAT (with associated SV40 small-t antigen sequence) is the reporter. In addition to the predicted transgene transcript (1.9 kb), we identified an abundant 1.5 kb transcript which derives from an alternative splicing event that utilises a cryptic splice donor site located within the CAT gene. The native CAT open reading frame (ORF) is lost in the 1.5 kb transcript, and a western analysis has shown that protein deriving from an aberrant open reading frame is not expressed at detectable levels.

Abstract: The arylalkylamine N-acetyltransferase (AA-NAT) gene is strongly expressed in the rat primarily in the pineal gland; low levels of expression are also found in the retina. AA-NAT catalyzes the key regulatory step controlling rhythmic melatonin output: the acetylation of serotonin. In the rat, the AA-NAT gene is expressed at night. This is controlled partly by cyclic AMP (cAMP) acting through a composite cAMP-responsive element-CCAAT site located upstream of the transcription start point. In the present study, we have extended our previous in vitro findings and found that additional elements in the 5' flanking region and first intron play an important role in the regulation of the AA-NAT gene. This led us to test the influence of an AA-NAT 5' flanking segment on the expression of the bacterial chloramphenicol acetyltransferase gene in a rat transgenic model. The results of this study clearly demonstrate that the segment of the AA-NAT gene that encompasses the minimal promoter and the first intron is able to confer the highly specific pineal/retinal and time-of-day patterns of AA-NAT gene expression. This advance also provides a tool that selectively targets genetic expression to pinealocytes and retinal photoreceptors, providing new experimental opportunities to probe gene expression in these tissues.

Abstract: This review cites new evidence suggesting a link between the recently discovered membrane bound water-selective channel, aquaporin-4 (AQP4), and the mechanism of central osmoreception. AQP4 is found in a number of brain regions associated with the osmoregulation of vasopressin secretion and thirst, including the supraoptic nucleus (SON) and subfornical organ (SFO). AQP4 expression is restricted to ependymal cell membranes in the SFO and astrocyte membranes in the SON, especially perivascular end foot processes, suggesting that glial cells may correspond to Verney's hypothalamic 'vesicular osmometers'. Information on osmotic status may thus be conveyed to the neuronal elements of the 'osmoreceptor complex' by a neurone-glial interaction.

Abstract: Exogenous GH inhibits endogenous GH release by hypothalamic feedback. We have recently exploited this to generate transgenic growth-retarded (Tgr) rats, in which human GH is expressed in the hypothalamus, under the control of the rat GRF gene promoter. These rats show reduced pituitary size, GH deficiency, and dominant dwarfism, but are large enough for serial blood sampling studies to examine their spontaneous GH secretion and responses to GRF, somatostatin, and GH-releasing peptide-6 (GHRP-6). Like their normal wild-type littermates, Tgr rats show a sexually dimorphic pattern of GH secretion; males secrete GH in 3-h episodes, whereas females exhibit a more continuous irregular output, with higher baseline GH levels. In anesthetized male Tgr rats, the GH responses to GRF or GHRP-6 were markedly reduced compared with those of their nontransgenic littermates, but the differences were smaller in females. Despite the reduction in pituitary GH, peak plasma GH responses to serial GRF injections in conscious Tgr males or intermittent somatostatin infusions in conscious Tgr females were indistinguishable from the responses in their wild-type littermates. Furthermore, 7-day iv infusions of GRF (12.5-100 micrograms/day), given either continuously or as a pulsatile infusion stimulated growth in Tgr rats, as did pulsatile infusions of GHRP-6. Thus, despite their pituitary GH deficiency and dwarfism, Tgr rats maintain a sexually dimorphic pattern of GH release and can produce large GH secretory responses to exogenous secretagogues. They represent the first genetic model of GH deficiency in the rat in which dwarfism can be corrected by treatment with exogenous GH secretagogues.

Abstract: Expression of human growth hormone (hGH) was targeted to growth hormone-releasing (GRF) neurons in the hypothalamus of transgenic rats. This induced dominant dwarfism by local feedback inhibition of GRF. One line, bearing a single copy of a GRF-hGH transgene, has been characterized in detail, and has been termed Tgr (for Transgenic growth-retarded). hGH was detected by immunocytochemistry in the brain, restricted to the median eminence of the hypothalamus. Low levels were also detected in the anterior pituitary gland by radioimmunoassay. Transgene expression in these sites was confirmed by RT-PCR. Tgr rats had reduced hypothalamic GRF and mRNA, in contrast to the increased GRF expression which accompanies GH deficiency in other dwarf rats. Endogenous GH mRNA, GH content, pituitary size and somatotroph cell number were also reduced significantly in Tgr rats. Pituitary adrenocorticotrophic hormone (ACTH) and thyroid-stimulating hormone (TSH) levels were normal, but prolactin content, mRNA levels and lactotroph cell numbers were also slightly reduced, probably due to feedback inhibition of prolactin by the lactogenic properties of the hGH transgene. This is the first dominant dwarf rat strain to be reported and will provide a valuable model for evaluating the effects of transgene expression on endogenous GH secretion, as well as the use of GH secretagogues for the treatment of dwarfism.

Abstract: Human GH (hGH) acts by dimerizing two hGH receptors that bind to different sites in hGH. G120RhGH, an analog mutated in the second binding site to prevent receptor dimerization, acts as an antagonist in vitro. We have now tested the activity of this analog in vivo in rats with low or absent endogenous GH secretion. Surprisingly, treatment with G120RhGH failed to antagonize the effects of infusions or injections of hGH in hypophysectomized (Hx) rats and had little effect on hepatic GH-sensitive CYP2C transcripts in GH-deficient dwarf (dw) rats. Paradoxically, G120RhGH stimulated skeletal growth when infused into Hx rats; a pulsatile iv infusion was more effective than a continuous pattern. Coinfusion of G120RhGH with hGH produced an additive effect on growth. In addition, continuous, but not pulsatile, G120RhGH infusion elevated hepatic 2C12 messenger RNA (mRNA) expression and reduced 2C11 mRNA expression in Hx rats. The direct effects of G120RhGH on hepatic CYP2C transcripts were confirmed in cultured hepatocytes in vitro, which also revealed a significant action of PRL in elevating 2C12 mRNA expression. Binding studies revealed that G120RhGH bound preferentially to hepatic PRL receptors, as [125I]G120hGH was completely displaced by ovine PRL but was unaffected by bGH, a specific GH receptor ligand. The weak growth-promoting effects of G120RhGH were similar to those induced by recombinant hPRL in Hx rats. Our results show that G120RhGH is a poor in vivo GH antagonist in the rat, but shows a paradoxical agonist effect, probably mediated by PRL receptors in this species.

Abstract: Quantitative 2-deoxyglucose (2-DG) autoradiography was employed to determine the effect of elevating cerebrospinal fluid osmolality on neuronal metabolism of hypothalamic periventricular regions in conscious rats. Injection of hypertonic mannitol solution into the dorsal third ventricle had no significant effect on local cerebral glucose utilization, whereas administration into the ventral third ventricle resulted in a significant elevation in 2-DG uptake in only the posterior pituitary, median preoptic nucleus (MnPo), median eminence and suprachiasmatic nucleus. These results indicate the contribution of a synaptic connection in the MnPo in the activation of the osmoreceptor complex.

Abstract: 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyses the reversible metabolism of corticosterone to inert 11-dehydrocorticosterone. At least two isoforms exist. 11 beta-HSD-1, the first to be characterised and the only isoform for which a cDNA has been isolated, is highly expressed in liver, kidney and hippocampus. The activity of 11 beta-HSD in rat liver is higher in males, due to oestrogen repression of 11 beta-HSD-1 gene transcription in females. Sexual dimorphism in rodent liver proteins is frequently mediated indirectly via sex-specific patterns of GH release (continuous in females, pulsatile in males). We have now investigated whether this applies to 11 beta-HSD, using dwarf rats (congenitally deficient in GH) and hypophysectomised animals. 11 beta-HSD activity and 11 beta-HSD-1 mRNA expression in liver was significantly lower in control female than male rats (50% and 72% of male levels respectively). These sex differences in the liver were attenuated in dwarf rats, with both males and females showing similar levels of 11 beta-HSD activity to control males. Administration of continuous (female pattern) GH to dwarf male rats decreased hepatic 11 beta-HSD activity (30% fall) and mRNA expression (77% fall), whereas the same total daily dose of GH given in the male (pulsatile) pattern had no effect on hepatic 11 beta-HSD in female dwarf rats. Continuous GH also attenuated hepatic 11 beta-HSD activity (25% fall) and 11 beta-HSD-1 mRNA expression (82% fall) in hypophysectomised animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Hepatic mRNA transcripts for the steroid-metabolizing enzymes cytochrome P4502C11 (male specific) and P4502C12 (female specific) differ in abundance by 10- to 20-fold in male and female rats and are regulated by their different patterns of GH secretion. This sex difference is also found in dwarf rats with low GH secretion, implying that these transcripts may be very sensitive to low level GH exposure. This has now been characterized in normal and dwarf rats. Continuous i.v. infusion of recombinant human (h) GH (0, 3, 12, and 48 micrograms/day) in both dwarf and normal male rats caused a dose-dependent decrease in P4502C11 and an increase in P4502C12, so that the 2C11/2C12 ratio fell from 17.9 +/- 1.3 to 1.5 +/- 1.0 in normal males and from 6.5 +/- 0.9 to 0.4 +/- 0.3 in dwarf males (0 vs. 48 micrograms hGH/day); over this dose range of hGH, body weight gain, total hepatic insulin-like growth factor-I mRNA levels, and plasma GHBP levels were largely unaffected. These effects of hGH were pattern dependent. The 2C11/2C12 ratio in dwarf males was feminized (from 11.9 +/- 1.3 to 0.08 +/- 0.03) by continuous infusion of hGH (36 micrograms/day), whereas a pulsatile infusion (3-min pulses every 3 h) of the same daily hGH dose was much less effective. Neither continuous nor pulsatile hGH affected P4502C11 or P4502C12 transcripts in dwarf females, although pulsatile hGH infusion caused a significant weight gain. To test whether baseline GH levels could be modified by circulating GH-binding protein (GHBP), hGH infusions were given with and without recombinant hGHBP in different patterns. Pulsatile infusions of recombinant hGHBP (42 micrograms/day, i.v.) did not prevent the feminizing effect of continuously infused hGH (36 micrograms/day, sc) in dwarf males (2C11/2C12 ratios were 0.08 +/- 0.01 and 0.09 +/- 0.01 for hGH vs. hGH plus hGHBP, respectively). This suggested that intermittent complex formation with GHBP did not prevent continuous access of hGH to the hepatic GH receptors. Furthermore, pulses of hGH complexed with GHBP significantly reduced the 2C11/2C12 ratio in dwarf males (from 21.5 +/- 3.9 with pulsatile hGH alone to 9.2 +/- 2.5 with pulses of hGH plus hGHBP), indicating that GHBP prolongs the exposure to hGH. Thus, 2C11/2C12 expression is very sensitive to basal GH levels in dwarf rats, and GHBP can alter hepatic gene expression by modifying the pattern of GH exposure.

Abstract: Salt and water balance and vasopressin secretion were measured in three colonies of Sprague-Dawley rats. Although sodium and water retention were similar between the groups, there were marked differences in both the rate and diurnal pattern of intake and excretion. Animals housed under semi-barrier conditions showed a lower basal plasma vasopressin concentration but were more sensitive to physiological stimuli. However, since pathogenic status and environmental conditions cannot entirely explain these results, genetic variation is likely to be a contributory factor.

Abstract: The effects of chronic and acute changes in plasma composition on the osmolality and sodium concentration of cerebrospinal fluid and plasma vasopressin (AVP) concentration have been examined. Chronic elevation of plasma osmolality in three strains of genetically AVP-deficient rats (Brattleboro and New Zealand hypertensive and normotensive Brattleboro) was associated with increased cerebrospinal fluid osmolality by comparison with AVP-replete controls (Long Evans and New Zealand genetically hypertensive and normotensive rats). The linear correlation between plasma and cerebrospinal fluid osmolality did not reflect a similar relationship between plasma and cerebrospinal fluid sodium concentration. Hypertensive animals exhibited a threefold higher plasma AVP concentration in association with significantly elevated cerebrospinal fluid osmolality by comparison with normotensive controls. Although ip hypertonic saline injection elicited parallel increases in plasma and cerebrospinal fluid osmolality and sodium concentration in both hypertensive and normotensive rats, only in the normotensives did this result in an increase in plasma AVP concentration. These results indicate that cerebrospinal fluid is subject to modest chronic and acute changes in osmolality and sodium concentration which may contribute to the osmotic control of AVP secretion. The disturbed control of vasopressin secretion in hypertensive rats may in part be related to the abnormal cerebrospinal fluid composition in these animals.

Abstract: The influence of aminergic pathways on basal and stimulated vasopressin (AVP) release was studied in conscious rats, the stimulus for hormone release being an intracerebroventricular (ICV) injection of 5 microliters 0.85M sodium chloride. The animals were treated with either phenoxybenzamine, propranolol or haloperidol prior to administration of the central hypertonic stimulus. Phenoxybenzamine elevated basal plasma vasopressin concentrations, while propranolol and haloperidol had no effect. The secretion of AVP in response to the hypertonic stimulus was potentiated by phenoxybenzamine and haloperidol, but the effect of propranolol was equivocal. The antagonists had no effect on basal arterial pressure at the time of hypertonic saline administration or the pressor response to ICV sodium chloride.

Abstract: There are GH-binding proteins (GHBPs) present in the blood of many species, and these correspond to the extracellular GH-binding domain of the GH receptor. In the rat, GHBP arises by alternative splicing of the GH receptor mRNA, but little is known of the physiological role of circulating GHBP, or its relationship with episodic GH secretion. We have developed a sensitive radioimmunoassay based on recombinant GHBP, and have measured rat GHBP levels in small samples of plasma from normal and GH-deficient dwarf rats. In normal adult rats, GHBP levels were two- to threefold higher in females than in males (16.6 +/- 0.8 vs 6.4 +/- 0.4 micrograms/l, P < 0.001), but this sex difference was not seen in dwarf rats. A continuous infusion of human GH in dwarf males raised plasma GHBP to 23.5 +/- 3.5 micrograms/l compared with 6.7 +/- 0.5 micrograms/l in sham-infused animals, whereas suppression of GH by continuous infusion of a long-acting somatostatin analogue in female dwarf rats had no effect on GHBP. In anaesthetized rats, large changes in plasma GH caused by i.v. administration of rat GH, somatostatin or GH-releasing factor did not affect GHBP acutely. Both GH and GHBP were also measured in serial blood samples from conscious normal and dwarf rats. A sexually dimorphic GH secretory pattern was observed in both strains. Males showed peaks and troughs of GH every 3 h varying over a 100-fold range, whereas females exhibited more continuous GH secretion. Despite the large fluctuations in endogenous GH, GHBP levels remained relatively constant in individual normal or dwarf males, as well as in females of both strains, and there was no significant correlation between GH and GHBP either in individual rats or as a group. Our results suggest that GHBP is GH-dependent in the longer term, and that the higher GHBP levels in female rats require their continuous GH secretory pattern. However, plasma GHBP levels remain stable and are not affected by acute changes in endogenous or exogenous GH.

Abstract: In rats, the onset of the sexually dimorphic pattern of growth hormone (GH) secretion and increased hepatic GH-binding capacity at puberty are temporally correlated with the sex-dependent expression of some hepatic cytochrome P450 enzymes involved in steroid metabolism. There are indications that the expression of the GH receptor gene itself is dependent on the sexually differentiated pattern of GH secretion. However, the molecular mechanisms by which a given pattern of GH secretion turns on a specific set of genes in the hepatocyte are not yet understood. Studies of the cytochrome P450 2C gene subfamily in hypophysectomized rats and isolated hepatocytes suggest that one major mechanism of GH action in the liver occurs through modulation of gene transcriptional initiation. The occurrence, in dwarf rats and in rats treated neonatally with monosodium glutamate, of sex differences in GH secretion and liver steroid metabolism typical of normal rats, in spite of a 95% reduction in pituitary GH levels, is compatible with the notion that extremely low levels of circulating GH are sufficient to regulate the expression of liver steroid-metabolizing enzymes. This, together with the fact that single daily subcutaneous injections of GH are sufficient to masculinize the liver of a hypophysectomized rat, indicates that neither the amplitude nor the frequency of the GH pulse is recognized as male or female by the hepatocyte, but rather the complete and prolonged suppression (in males) or the persistence (in females) of circulating GH during the trough period after a GH surge.

Abstract: A series of studies has been performed in the conscious rat to investigate the effect of the intracerebroventricular (i.c.v.) administration of the selective kappa-opioid receptor agonist, U50 488H, on arginine vasopressin (AVP) secretion stimulated by i.c.v. administration of hypertonic NaCl. Similarly, the effect of the i.c.v. administration of morphine and the i.v. administration of naloxone on AVP secretion was investigated. The response of AVP to an i.c.v. injection of hypertonic NaCl was potentiated by naloxone at a dose of 0.4 mg/kg, but a higher dose (1.2 mg/kg) was required to increase the basal plasma concentration of AVP. Prior treatment with U50 488H or morphine attenuated the increase in plasma concentrations of AVP stimulated by i.c.v. injection of hypertonic NaCl from 13.92 +/- 4.44 to 1.22 +/- 0.34 and 1.78 +/- 0.74 pmol/l respectively (n = 7; P less than 0.05). Prior administration of U50 488H also attenuated the potentiating effect of naloxone on AVP secretion stimulated by i.c.v. injection of hypertonic NaCl. These results indicate that basal AVP secretion is under tonic inhibitory control by dynorphin, and that mu- and kappa-opioid receptors mediate an inhibitory influence of endogenous opioids on osmoreceptor-mediated AVP secretion.

Abstract: 1. Intracerebroventricular (I.C.V.) injections of isotonic and hypertonic solutions into the dorsal (D3V) and ventral (V3V) third ventricle were employed to examine the release of vasopressin (AVP) and the mean arterial pressure (MAP) response to elevated cerebrospinal fluid (CSF) osmolality in the conscious rat. 2. The D3V injection of hypertonic sodium chloride solution was associated with a concentration-dependent, transient increase in plasma AVP concentration and MAP. 3. The D3V injection of 5 microliters 0.85 M-sodium chloride elicited a 7-fold increase in plasma AVP and oxytocin concentrations, but had no effect on plasma ACTH concentration. The D3V injection of 1.11 M-mannitol in 0.15 M-sodium chloride had no effect on plasma AVP concentration or MAP. However, the D3V injection of 0.746 M-mannitol in 0.4 M-sodium chloride elicited a significant transient increase in plasma AVP, but had no effect on MAP. 4. The V3V injection of 5 microliters 0.85 M-sodium chloride elicited a prolonged increase in plasma AVP concentration and a transient increase in MAP. The V3V injection of 5 microliters 1.11 M-mannitol in 0.15 M-sodium chloride elicited an equal, but transient, increase in plasma AVP concentration, but had no effect on MAP. 5. The pressor effect of a D3V injection of 0.85 M-sodium chloride was unaffected by prior administration of the V1 (pressor) receptor antagonist (beta-mercapto-beta,beta-cyclopentamethylene propionyl1, O-Me-Tyr2, Arg8)-vasopressin. 6. These results indicate that osmotically induced AVP secretion may be mediated by both sodium receptors and osmoreceptors, although expression of the response may depend upon the maintenance of a 'permissive' concentration of sodium in the CSF. 7. It appears also that the pressor effect is not due to increased plasma AVP concentration, but only results from elevation of the CSF sodium concentration.