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Nutrition plays a critical role in programming and shaping linear growth during early postnatal life through direct action on the development of the neuroendocrine somatotropic (GH/IGF-1) axis. IGF-1 is a key factor in modulating the programming of linear growth during this period. Notably, IGF-1 preferentially stimulates axonal growth of GHRH neurons in the arcuate nucleus of the hypothalamus (Arc), which is crucial for the proliferation of somatotroph progenitors in the pituitary, thus influencing later GH secretory capacity. However, other nutrition-related hormones may also be involved. Among them, insulin shares several structural and functional similarities with IGF-1, as well as downstream signaling effectors. We investigated the role of insulin in the control of Arc axonal growth using an in vitro model of arcuate explants culture and a cell-type specific approach (GHRH-eGFP mice) under both physiological conditions (normally fed pups) and those of dietary restriction (underfed pups). Our data suggest that insulin failed to directly control axonal growth of Arc neurons or influence specific IGF-1-mediated effects on GHRH neurons. Insulin may act on neuronal welfare, which appears to be dependent on neuronal sub-populations and is influenced by the nutritional status of pups in which Arc neurons develop.

International audience; Pancreatic β-cells modulate insulin secretion through rapid sensing of blood glucose and integration of gut-derived signals. Increased insulin demand during pregnancy and obesity alters islet function and mass and leads to gestational diabetes mellitus and type 2 diabetes in predisposed individuals. However, it is unclear how blood-borne factors dynamically access the islets of Langerhans. Thus, understanding the changes in circulating molecule distribution that accompany compensatory β-cell expansion may be key to developing novel antidiabetic therapies. Here, using two-photon microscopy in vivo in mice, we demonstrate that islets are almost instantly exposed to peaks of circulating molecules, which rapidly pervade the tissue before clearance. In addition, both gestation and short-term high-fat-diet feeding decrease molecule extravasation and uptake rates in vivo in islets, independently of β-cell expansion or islet blood flow velocity. Together, these data support a role for islet vascular permeability in shaping β-cell adaptive responses to metabolic demand by modulating the access and sensing of circulating molecules.

International audience; Nutrition during the perinatal period programs body growth. Growth hormone (GH) secretion from the pituitary regulates body growth and is controlled by Growth Hormone Releasing Hormone (GHRH) neurons located in the arcuate nucleus of the hypothalamus. We observed that dietary restriction during the early postnatal period (i.e. lactation) in mice influences postnatal growth by permanently altering the development of the somatotropic axis in the pituitary gland. This alteration may be due to a lack of GHRH signaling during this critical developmental period. Indeed, underfed pups showed decreased insulin-like growth factor I (IGF-I) plasma levels, which are associated with lower innervation of the median eminence by GHRH axons at 10 days of age relative to normally fed pups. IGF-I preferentially stimulated axon elongation of GHRH neurons in in vitro arcuate explant cultures from 7 day-old normally fed pups. This IGF-I stimulating effect was selective since other arcuate neurons visualized concomitantly by neurofilament labeling, or AgRP immunochemistry, did not significantly respond to IGF-I stimulation. Moreover, GHRH neurons in explants from age-matched underfed pups lost the capacity to respond to IGF-I stimulation. Molecular analyses indicated that nutritional restriction was associated with impaired activation of AKT. These results highlight a role for IGF-I in axon elongation that appears to be cell selective and participates in the complex cellular mechanisms that link underfeeding during the early postna-tal period with programming of the growth trajectory.

[This corrects the article DOI: 10.1371/journal.pone.0170083.].

Cities today have two major environmental concerns – carbon emissions and air quality. Global carbon levels are increasing and cities require to show plans to tackle and reduce the amount of carbon which they are emitting. At present carbon emissions in urban areas are calculated rather than measured. In some cities where industrial activity is not carbon intensive, the major contributors are the burning of fuel for heating and the emissions from vehicles. Air quality levels have a direct impact on human health and cities are under increased pressure to demonstrate plans to control and reduce levels of air pollution. Of great importance is the way in which emissions, both carbon rich emissions and pollutants, disperse in a city environment. Little work has been reported on the movement of CO2 in the urban environment and the effect the structure of the environment exerts on the movement and dispersion. This paper describes an investigation into the dispersion of CO2 within an urban environment in the Old Town of the City of Edinburgh, using a hand carried low cost portable CO2 sensor.

Summary. objective To study the regulation of the growth hormone (GH) response to growth hormone releasing hormone (GHRH) in the presence or absence of somatostatin pretreatment. design Seven healthy adult male volunteers of normal height and weight and aged between 19 and 29 years underwent four separate studies (each containing three cycles In one day) In random order. The studies were separated from each other by at least a week. On day 1, three consecutive cycles (between 0800 and 2000 hours) consisted each of a saline infusion for 3 hours which was stopped prior to a bolus Injection of saline and followed by 60 minutes of more intensive blood sampling. On day 2, the bolus Injections were of GHRH given after saline infusion. On days 3 and 4 somatostatin Infusions were administered instead of saline over the 3-hour periods followed by bolus injections of saline or GHRH respectively. In all studies, samples were collected for the measurement of serum GH concentration at 15-minute Intervals from time 0 to 180 minutes and then at 5-minute Intervals for a further 60 minutes, this cycle being repeated three times. measurements Serum GH concentrations were analysed by serial array averaging. results Prompt release of GH was observed In response to GHRH given against a saline background (day 2, cycle 1) (mean at 60 minutes 49.2±14.7 mU/1) but the responses observed during the second and third cycles were attenuated (mean at 60 minutes 17.2±4.0 mU/1; p=0025). GH release between somatostatin infusions (somatostatin withdrawal; day 3) occurred twice as often as that observed during saline Infusions (62% day 3: 29% day 1). The response, although qualitatively similar to that Induced by GHRH, was reduced in amplitude and the time of onset variable (5–45 minutes). On day 4, the administration of GHRH as a bolus injection combined with somatostatin withdrawal led to consistent and repeatable GH responses (mean at 60 minutes, cycle 1, 39.7±10.8 mU/1; cycles 2 and 3, 37.4±9.4 mU/1) which were similar to those observed with GHRH alone (day 2, cycle 1) (mean 39.7±10.8 mU/1) (p=NS). conclusions These data suggest that endogenous somatostatin secretion is important In determining the ability of the somatotroph to respond to repeated growth hormone releasing hormone stimulation and that for regular GH pulse generation a close Interplay between growth hormone releasing hormone and somatostatin is required.

Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K+ channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These “feed-forward loops” may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.

The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size. However, cell motility, a process commonly observed in a variety of tissues during development, may represent an additional mechanism to promote plasticity within the adult pituitary gland. To investigate this, we used multiphoton time-lapse imaging methods, GH-enhanced green fluorescent protein transgenic mice and sexual dimorphism of the GH axis as a model of divergent tissue demand. Using these methods to acutely (12 h) track cell dynamics, we report that ovariectomy induces a dramatic and dynamic increase in cell motility, which is associated with gross GH-cell network remodeling. These changes can be prevented by estradiol supplementation and are associated with enhanced network connectivity as evidenced by increased coordinated GH-cell activity during multicellular calcium recordings. Furthermore, cell motility appears to be sex-specific, because reciprocal alterations are not detected in males after castration. Therefore, GH-cell motility appears to play an important role in the structural and functional pituitary plasticity, which is evoked in response to changing estradiol concentrations in the female.

GH secretion and growth rates are developmentally regulated and sexually dimorphic, but the neuroregulatory mechanisms between birth and puberty are unclear. Using the GHRH-enhanced green fluorescent protein (eGFP) transgenic mouse, in which eGFP provides a strong surrogate signal for identifying GHRH neurons, we showed that numbers in the male arcuate nucleus were double those seen in females at x postnatal day (P)1 and P10, during which time numbers increased 2- to 3-fold. Thereafter (P20, P30, P60, P365) there was a significant trend for numbers to decrease in males and increase in females, such that sex differences were, surprisingly, absent in young and late adulthood. Conversely, we identified the emergence of male-dominant sex differences in the number of processes extended per GHRH perikarya across puberty. Intriguingly, prepubertal gonadectomy (P28), unlike adult gonadectomy, caused a dramatic 40% loss of GHRH cells in both sexes in adulthood and a significant (30%) increase in processes emanating from cell bodies only in females. These findings establish a novel ontogenetic profile for GHRH neurons and suggest previously undiscovered roles for peripubertal gonadal factors in establishing population size in both sexes. They also provide the first demonstration of emergent sex-specific GHRH architecture, which may signal the onset of sex-dependent regulation of activity reported for adult GHRH-eGFP neurons, and its differential regulation by gonadal factors in males and females. This information adds to our knowledge of processes that underpin the emergence of sex-specific GH secretory dynamics and hence biological activity of this pleiotropic hormone.

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

Isolated GH deficiency type II (IGHD II) is the autosomal dominant form of GHD. In the majority of the cases, this disorder is due to specific GH-1 gene mutations that lead to mRNA missplicing and subsequent loss of exon 3 sequences. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH (Delta3GH) isoform that reduces the accumulation and secretion of wild-type-GH. At present, patients suffering from this type of disease are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the Delta3GH mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. We developed a strategy involving Delta3GH isoform knockdown mediated by expression of a microRNA-30-adapted short hairpin RNA (shRNA) specifically targeting the Delta3GH mRNA of human (shRNAmir-Delta3). Rat pituitary tumor GC cells expressing Delta3GH upon doxycycline induction were transduced with shRNAmir-Delta3 lentiviral vectors, which significantly reduced Delta3GH protein levels and improved human wild-type-GH secretion in comparison with a shRNAmir targeting a scrambled sequence. No toxicity due to shRNAmir expression could be observed in cell proliferation assays. Confocal microscopy strongly suggested that shRNAmir-Delta3 enabled the recovery of GH granule storage and secretory capacity. These viral vectors have shown their ability to stably integrate, express shRNAmir, and rescue IGHD II phenotype in rat pituitary tumor GC cells, a methodology that opens new perspectives for the development of gene therapy to treat IGHD patients.

Background/Aims. 20 Kilodalton-hGH (20 K-hGH) is the second most abundant pituitary GH variant after 22 K-hGH. In the steady state the proportion of 20 : 22 K-hGH appears constant; does this proportion change with repetitive somatotroph stimulation? Methods. Forty adult males were randomised to receive a GHRH(1–29) bolus ( or ( , preceded or followed by a saline bolus, 1 week apart. Four to six weeks later, 10 subjects received GHRH(1–29) at 0, 60, 120, and 180 minutes. Clearance rate of 22 and 20 K-hGH was measured in 10 subjects. Results. Total amount/proportion of 22 K-hGH/20 K-hGH secreted was similar for both GHRH(1–29) doses. Repetitive stimulation reduced the amount of 22 K-hGH released whereas the amount of 20 K-hGH did not change significantly leading to an increase in the proportion of 20 K-hGH . Half-life of 20 and 22 K-hGH were not significantly different . Conclusions. Repetitive stimulation of the somatotroph may alter the proportion of GH variant released.

Our view of anterior pituitary organization has been altered with the recognition that folliculo-stellate (FS) and somatotroph cell populations form large-scale three-dimensional homotypic networks. This morphological cellular organization may optimize communication within the pituitary gland promoting coordinated pulsatile secretion adapted to physiological needs. The aim of this study was to identify the molecules involved in the formation and potential functional organization and/or signaling within these cell–cell networks. Here, we have focused on one class of cell adhesion molecules, the cadherins, since β-catenin has been detected in the GH cell network. We have characterized, by qPCR and immunohistochemistry, their cellular expression and distribution. We have also examined whether their expression could be modulated during pituitary tissue remodeling. The mouse anterior pituitary has a restricted and cell-type specific repertoire of cadherin expression: cadherin-11 is exclusively expressed in TSH cells; N-cadherin displays a ubiquitous expression pattern but with different levels of expression between endocrine cell types; E-cadherin is restricted to homotypic contacts between FS cells; while cadherin-18 is expressed both in somatotrophs and FS cells. Thus, each cell type presents a defined combinatorial expression of different subsets of cadherins. This cell-type specific cadherin expression profile emerges early during development and undergoes major changes during postnatal development. These results suggest the existence within the anterior pituitary of cell–cell contact signaling based on a defined pattern of cadherin expression, which may play a crucial role in cellular recognition during the formation and fate of pituitary cell homotypic networks.

International audience; In mammals, males and females exhibit anatomical, hormonal, and metabolic differences. A major example of such sex dimorphism in mouse involves hepatic drug metabolism, which is also a noticeable target of circadian timekeeping. However, whether the circadian clock itself contributes to sex-biased metabolism has remained unknown, although several daily output parameters differ between sexes in a number of species, including humans. Here we show that dimorphic liver metabolism is altered when the circadian regulators Cryptochromes, Cry1 and Cry2, are inactivated. Indeed, double mutant Cry1(-/-) Cry2(-/-) male mice that lack a functional circadian clock express a number of sex-specific liver products, including several cytochrome P450 enzymes, at levels close to those measured in females. In addition, body growth of Cry-deficient mice is impaired, also in a sex-biased manner, and this phenotype goes along with an altered pattern of circulating growth hormone (GH) in mutant males, specifically. It is noteworthy that hormonal injections able to mimic male GH pulses reversed the feminized gene expression profile in the liver of Cry1(-/-) Cry2(-/-) males. Altogether, our observations suggest that the 24-h clock paces the dimorphic ultradian pulsatility of GH that is responsible for sex-dependent liver activity. We thus conclude that circadian timing, sex dimorphism, and liver metabolism are finely interconnected.

Context: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus.Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues.Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke’s pouch, the developing anterior pituitary, and the eye.Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

Transgenic Late-onset OBesity (LOB) rats slowly develop a male-specific, autosomal dominant, obesity phenotype with a specific increase in peri-renal white adipose tissue (WAT) depot and preserved insulin sensitivity (Bains et al. in Endocrinology 145:2666-2679, 2004). To better understand the remarkable phenotype of these rats, the lipid metabolism was investigated in male LOB and non-transgenic (NT) littermates. Total plasma cholesterol (C) levels were normal but total plasma triacylglycerol (TAG) (2.8-fold) and hepatic TAG content (25%) was elevated in LOB males. Plasma VLDL-C and VLDL-TAG levels were higher while plasma apoB levels were 60% lower in LOB males. Increased hepatic TAG secretion explained the increased VLDL levels in LOB males. The hepatic gene expression of FAS, SCD-1, mitochondrial (mt)GPAT, and DGAT2 was up-regulated in both old obese and young non-obese LOB rats. Lipoprotein lipase (LPL) activity in heart and epididymal white adipose tissue (WAT) was unchanged, while LPL activity was increased in peri-renal WAT (30%) and decreased in soleus muscle (40%). Moreover, FAS, SCD-1 and DGAT2 gene expression was increased in peri-renal, but not in epididymal WAT. Basal lipolysis was reduced or unchanged and beta-adrenergic stimulated lipolysis was reduced in WAT from both old obese and young non-obese LOB rats. To summarize, the obese phenotype of LOB male rats is associated with increased hepatic TAG production and secretion, a shift in LPL activity from skeletal muscle to WAT, reduced lipolytic response in WAT depots and a specific increase in expression of genes responsible for fatty acid and TAG synthesis in the peri-renal depot.

The pituitary gland adapts the proportion of each of its endocrine cell types to meet differing hormonal demands throughout life. There is circumstantial evidence that multipotent adult progenitor cells contribute to this plasticity, but these cells have not been identified. Here, we describe a small (+ cells in the adult gland also express SOX9 and S100. We suggest that this SOX2 + /SOX9 + population represents transit-amplifying cells, whereas the SOX2 + /SOX9 − cells we identify are multipotent progenitor/stem cells persisting in the adult pituitary.

Context and Objective: Alteration of exon splice enhancers (ESE) may cause autosomal dominant GH deficiency (IGHD II). Disruption analysis of a (GAA) (n) ESE motif within exon 3 by introducing single-base mutations has shown that single nucleotide mutations within ESE1 affect pre-mRNA splicing.Design, Setting, and Patients: Confirming the laboratory-derived data, a heterozygous splice enhancer mutation in exon 3 (exon 3 + 2 A→C) coding for GH-E32A mutation of the GH-1 gene was found in two independent pedigrees, causing familial IGHD II. Because different ESE mutations have a variable impact on splicing of exon 3 of GH and therefore on the expression of the 17.5-kDa GH mutant form, the GH-E32A was studied at the cellular level.Interventions and Results: The splicing of GH-E32A, assessed at the protein level, produced significantly increased amounts of 17.5-kDa GH isoform (55% of total GH protein) when compared with the wt-GH. AtT-20 cells coexpressing both wt-GH and GH-E32A presented a significant reduction in cell proliferation as well as GH production after forskolin stimulation when compared with the cells expressing wt-GH. These results were complemented with confocal microscopy analysis, which revealed a significant reduction of the GH-E32A-derived isoform colocalized with secretory granules, compared with wt-GH.Conclusion: GH-E32A mutation found within ESE1 weakens recognition of exon 3 directly, and therefore, an increased production of the exon 3-skipped 17.5-kDa GH isoform in relation to the 22-kDa, wt-GH isoform was found. The GH-E32A mutant altered stimulated GH production as well as cell proliferation, causing IGHD II.

Recent evidence suggests that alterations in insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) can increase mammalian life span. For example, in several mouse mutants, impairment of the growth hormone (GH)/IGF1 axis increases life span and also insulin sensitivity. However, the intracellular signaling route to altered mammalian aging remains unclear. We therefore measured the life span of mice lacking either insulin receptor substrate (IRS) 1 or 2, the major intracellular effectors of the IIS receptors. Our provisional results indicate that female Irs1-/- mice are long-lived. Furthermore, they displayed resistance to a range of age-sensitive markers of aging including skin, bone, immune, and motor dysfunction. These improvements in health were seen despite mild, lifelong insulin resistance. Thus, enhanced insulin sensitivity is not a prerequisite for IIS mutant longevity. Irs1-/- female mice also displayed normal anterior pituitary function, distinguishing them from long-lived somatotrophic axis mutants. In contrast, Irs2-/- mice were short-lived, whereas Irs1+/- and Irs2+/- mice of both sexes showed normal life spans. Our results therefore suggest that IRS1 signaling is an evolutionarily conserved pathway regulating mammalian life span and may be a point of intervention for therapies with the potential to delay age-related processes.

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.

In the present work, we took advantage of a recently described model of GHRH-enhanced green fluorescent protein (eGFP) transgenic mice to evaluate the extent of co-localization of GHRH neurons with galanin (GAL), neurotensin (NT) and tyrosine hydroxylase (TH) in 3- and 8-month-old male and female mice. The total number of GHRH-eGFP neurons along the rostro-caudal axis of the arcuate nucleus did not differ according to gender or age. GAL-immunoreactivity was present in 40–44% of 3-month-old GHRH-eGFP neurons in male and female arcuate nucleus, respectively, but only 25–22% in 8-month-old mice. TH immunoreactivity occurred in 36–35% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice and these proportions increased to 40 and 45% in 8-month-old mice. NT immunoreactivity was present in 14 and 24% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice up to 28 and 26% in 8-month-old mice. Thus, co-localization of peptides and enzyme in GHRH-eGFP neurons displays a sexual dimorphism at 3-month of age for NT, and at 8-month for TH, while the total number of GHRH-eGFP neurons does not exhibit gender difference at either age. In summary, it appears that changes in co-localized (and presumably co-released) peptides, rather than GHRH per se , may contribute to the changes in sexually dimorphic GH secretion with aging in the mouse.

We report the development of a differential absorption lidar instrument (DIAL) designed and built specifically for the measurement of anthropogenic greenhouse gases in the atmosphere. The DIAL is integrated into a commercial astronomical telescope to provide high-quality receiver optics and enable automated scanning for three-dimensional lidar acquisition. The instrument is portable and can be set up within a few hours in the field.The laser source is a pulsed optical parametric oscillator (OPO) which outputs light at a wavelength tunable near 1.6 μm. This wavelength region, which is also used in telecommunications devices, provides access to absorption lines in both carbon dioxide at 1573 nm and methane at 1646 nm. To achieve the critical temperature stability required for a laser- based field instrument the four-mirror OPO cavity is machined from a single aluminium block. A piezoactuator adjusts the cavity length to achieve resonance and this is maintained over temperature changes through the use of a feedback loop. The laser output is continuously monitored with pyroelectric detectors and a custom-built wavemeter.The OPO is injection seeded by a temperature-stabilized distributed feedback laser diode (DFB-LD) with a wavelength locked to the absorption line centre (on-line) using a gas cell containing pure carbon dioxide. A second DFB-LD is tuned to a nearby wavelength (off-line) to provide the reference required for differential absorption measurements. A similar system has been designed and built to provide the injection seeding wavelengths for methane. The system integrates the DFB-LDs, drivers, locking electronics, gas cell and balanced photodetectors.The results of test measurements of carbon dioxide are presented and the development of the system is discussed, including the adaptation required for the measurement of methane.

From the early scanning spectrometers, the utility field spectroscopy has been constrained: by detector sensitivity, leading to high integration times; portability; and the measurements having very limited support (possibly an Earth surface area in the order of 0.25 square metres to 2 square metres). However, over the last twenty years or so detector sensitivities and electronics have in improved leading to practical Earth surface sampling time increasing their utility to support Earth observation science and as optical remote sensing teaching and training tools. Now the uncertainties associated with field spectral measurements are being more widely recognised and field sampling methods and instrument continue to be developed to enable these uncertainties to be quantified and minimised. There are a number of key challenges which still need to be more widely addressed if field spectroscopy is to provide evermore reliable and replicable measurements. An understanding of the systematic biases introduced by this sampling method has begun to be recognised. In addition, the mismatch in scale between near-ground spectroscopy measurement and observations from space-borne sensor has begun to be addressed with the development of unmanned aerial vehicles as platforms and lightweight and miniaturised state- of-the-art spectrometer systems. It is now possible to non-invasively sample terrestrial and hydrological ecosystems in a statistically robust manner and do so with supports similar in scale to those of air- and space-borne sensors. These developments will revolutionise the use of field spectroscopy to support empirical science and model development and the calibration and validation of space-based observations.

Muscle cells, like almost all other cells in the body, show growth and metabolic responses to growth hormone (GH). Although GH actions are classically thought to be mediated via insulin-like growth factor I (IGF-I), it is now recognized that GH has direct actions via the GH receptor (GHR) expressed in many tissues, including muscle. The aim of this work was to study, at an individual cell and tissue level, those cells that respond directly to GH, and understand how their responsiveness to GH is regulated. We used immunocytochemistry to visualize the cellular expression of components of the major GH signal transduction cascades, GHR, Janus kinase 2 (JAK2), signal transducer and activator of transcription 5b (Stat5b) and suppressor of cytokine signalling 2 (SOCS2), and monitored their activation by detecting GH-induced phosphorylation after a single injection of GH in GH-deficient rodents. GHR, JAK2, Stat5b and SOCS2 proteins were all found to be present in skeletal muscle from normal and GH-deficient mice, and phosphorylated Stat5b increased in heart and skeletal muscle nuclei after a GH injection. In GH-deficient rats, continuous GH infusion increased Stat5b phosphorylation in liver but not in muscle, but blunted the further effects on Stat5b of acute GH injection in both tissues. Extracellular signal-regulated kinase (ERK), SOCS2 and cytokine-inducible SH2 domain-containing protein (CIS) are also activated by GH in different tissues, and other studies have implicated cross-talk with insulin/IGF-I transduction pathways. Activation of several of these pathways could be traced using these methods, which will shed light on the molecular mechanisms that regulate GH responsiveness in individual cells in complex tissues.

Pituitary growth hormone (GH)-secreting cells regulate growth and metabolism in animals and humans. To secrete highly ordered GH pulses (up to 1,000-fold rise in hormone levels in vivo ), the pituitary GH cell population needs to mount coordinated responses to GH secretagogues, yet GH cells display an apparently heterogeneous scattered distribution in 2D histological studies. To address this paradox, we analyzed in 3D both positioning and signaling of GH cells using reconstructive, two-photon excitation microscopy to image the entire pituitary in GH-EGFP transgenic mice. Our results unveiled a homologous continuum of GH cells connected by adherens junctions that wired the whole gland and exhibited the three primary features of biological networks: robustness of architecture across lifespan, modularity correlated with pituitary GH contents and body growth, and connectivity with spatially stereotyped motifs of cell synchronization coordinating cell activity. These findings change our view of GH cells, from a collection of dispersed cells to a geometrically connected homotypic network of cells whose local morphology and connectivity can vary, to alter the timing of cellular responses to promote more coordinated pulsatile secretion. This large-scale 3D view of cell functioning provides a powerful approach to identify and understand other networks of endocrine cells that are thought to be scattered in situ . Many dispersed endocrine systems exhibit pulsatile outputs. We suggest that cell positioning and associated cell-cell connection mechanisms will be critical parameters that determine how well such systems can deliver a coordinated secretory pulse of hormone to their target tissues.

Duplications of Xq26-27 have been implicated in the etiology of X-linked hypopituitarism associated with mental retardation (MR). Additionally, an expansion of a polyalanine tract (by 11 alanines) within the transcription factor SOX3 (Xq27.1) has been reported in patients with growth hormone deficiency and variable learning difficulties. We report a submicroscopic duplication of Xq27.1, the smallest reported to date (685.6 kb), in two siblings with variable hypopituitarism, callosal abnormalities, anterior pituitary hypoplasia (APH), an ectopic posterior pituitary (EPP), and an absent infundibulum. This duplication contains SOX3 and sequences corresponding to two transcripts of unknown function; only Sox3 is expressed in the infundibulum in mice. Next, we identified a novel seven-alanine expansion within a polyalanine tract in SOX3 in a family with panhypopituitarism in three male siblings with an absent infundibulum, severe APH, and EPP. This mutation led to reduced transcriptional activity, with impaired nuclear localization of the mutant protein. We also identified a novel polymorphism (A43T) in SOX3 in another child with hypopituitarism. In contrast to findings in previous studies, there was no evidence of MR or learning difficulties in our patients. We conclude that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily MR.

The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.

Isolated growth hormone deficiency type II (IGHD II) is characterized by short stature due to dominant-negative mutations of the human growth hormone gene (GH1). Most of the known mutations responsible for IGHD II cause aberrant splicing of GH1 transcripts. We have recently shown that mutations that cause exon 3 skipping and produce a dominant-negative 17.5-kDa isoform in humans also cause a dose-dependent disruption of GH secretory vesicles when expressed in GC cells and transgenic mice. We show here that overexpression of the dominant-negative 17.5-kDa isoform also destroys the majority of somatotrophs, leading to anterior pituitary hypoplasia in transgenic mice. It is, therefore, important to understand the regulation of GH1 splicing and why its perturbation causes IGHD II. We demonstrate that dual splicing enhancers are required to ensure exon 3 definition to produce full-length 22-kDa hormone. We also show that splicing enhancer mutations that weaken exon 3 recognition produce variable amounts of the 17.5-kDa isoform, a result which could potentially explain the clinical variability observed in IGHD II. Non-canonical splicing mutations that disrupt splicing enhancers, such as those illustrated here, demonstrate the importance of enhancer elements in regulating alternative splicing to prevent human disease.

Autosomal dominant GH deficiency type II (IGHDII) is often associated with mutations in the human GH gene (GH1) that give rise to products lacking exon-3 ((Deltaexon3)hGH). In the heterozygous state, these act as dominant negative mutations that prevent the release of human pituitary GH (hGH). To determine the mechanisms of these dominant negative effects, we used a combination of transgenic and morphological approaches in both in vitro and in vivo models. Rat GC cell lines were generated expressing either wild-type GH1 (WT-hGH-GC) or a genomic GH1 sequence containing a G->A transition at the donor splice site of IVS3 ((Deltaexon3)hGH-GC). WT-hGH-GC cells grew normally and produced equivalent amounts of human and rGH packaged in dense-cored secretory vesicles (SVs). In contrast, (Deltaexon3)hGH-GC cells showed few SVs but accumulated secretory product in amorphous cytoplasmic aggregates. They produced much less rGH and grew more slowly than WT-hGH-GC cells. When cotransfected with an enhanced green fluorescent protein construct (GH-eGFP), which copackages with GH in SVs, WT-hGH-GC cells showed normal electron microscopy morphology and SV movements, tracked with total internal reflectance fluorescence microscopy. In contrast, coexpression of (Deltaexon3)hGH with GH-eGFP abolished the vesicular targeting of GH-eGFP, which instead accumulated in static aggregates. Transgenic mice expressing (Deltaexon3)hGH in somatotrophs showed an IGHD-II phenotype with mild to severe pituitary hypoplasia and dwarfism, evident at weaning in the most severely affected lines. Hypothalamic GHRH expression was up-regulated and somatostatin expression reduced in (Deltaexon3)hGH transgenic mice, consistent with their profound GHD. Few SVs were detectable in the residual pituitary somatotrophs in (Deltaexon3)hGH transgenic mice, and these cells showed grossly abnormal morphology. A low copy number transgenic line showed a mild effect relatively specific for GH, whereas two severely affected lines with higher transgene copy numbers showed early onset, widespread pituitary damage, macrophage invasion, and multiple hormone deficiencies. These new in vitro and in vivo models shed new light on the cellular mechanisms involved in IGHDII, as well as its phenotypic consequences in vivo.

Bone marrow (BM) contains numerous adipocytes. These share a common precursor with osteoblasts and chondrocytes, but their function is unknown. It is unclear what regulates the differentiation of these three different cell types, though their subsequent metabolic activity is under hormonal regulation. GH and estrogen stimulate bone growth and mineralization, by direct effects on chondrocytes and osteoblasts. GH also stimulates lipolysis in subcutaneous and visceral adipocytes. However, adipocytes in BM have largely been ignored as potential targets for GH or estrogen action. We have addressed this by measuring BM adipocyte number, perimeter and area as well as bone area and osteoblast activity in GH-deficient dwarf (dw/dw), normal, or ovariectomized (Ovx) rats, with or without GH, IGF-1, PTH, or estrogen treatment or high fat feeding. Marrow adipocyte numbers were increased 5-fold (P < 0.001) in dw/dw rats, and cell size was also increased by 20%. These values returned toward normal in dw/dw rats given GH but not when given IGF-1. Cancellous bone area and osteoblast number were significantly (P < 0.005) lower in dw/dw rats, though alkaline phosphatase (ALP) activity in individual osteoblasts was unchanged. GH treatment increased % osteoblast covered bone surface without affecting individual cell ALP activity. Ovariectomy in normal or dw/dw rats had no affect on marrow adipocyte number nor size, although estrogen treatment in ovariectomized (Ovx) normal rats did increase adipocyte number. Ovx decreased tibial cancellous bone area in normal rats (64%; P < 0.05) and decreased osteoblast ALP-activity (P < 0.01) but did not affect the percentage of osteoblast-covered bone surface. Estrogen replacement reversed these changes. While treatment with PTH by continuous sc infusion decreased cancellous bone (P < 0.05) and high fat feeding increased the size of BM adipocytes (P < 0.01), they did not affect BM adipocyte number. These results suggest that GH has a specific action on BM adipocytes that is not simply due to altered bone or fat metabolism. We conclude that the marrow adipocyte lineage is an important and specific target for GH action. The inverse relationship between adipocyte number and osteoblast covered bone surface, together with the well-known effects of GH on epiphysial chondrocytes leads us to propose that GH plays two important roles on cells of all three lineages. During differentiation, it regulates the numbers of each cell type that are maintained from the common precursor lineage. Subsequently it has cell-specific effects on the metabolic activities of the differentiated cells. In the case of marrow adipocytes, GH-dependent lipolysis could provide an important hormonally regulated local high energy source in bone. (Endocrinology 143: 4065– 4073, 2002)

Cranial irradiation in children and adults often results in irreversible hypopituitarism. The earliest and most common endocrine abnormality is GH deficiency, often followed by other pituitary hormone deficits. We investigated whether a similar pattern of progressive hypopituitarism could be reproduced in an animal model. Different doses of cranial irradiation were delivered to the hypothalamo-pituitary region of normal adult male rats, and the effects on their subsequent growth, pituitary weight and hormone contents were studied. Animals received cranial irradiation with 300 kV X-rays at doses of 0, 20, 22 or 24 Gy (n=15 per group) and five animals from each group were killed at 8, 14 or 20 weeks after irradiation. Their anterior pituitary glands were weighed and assayed for GH, LH, TSH, ACTH and prolactin (PRL) content. All three doses of irradiation reduced body weight compared with that in non-irradiated controls and compromised growth between 8 and 20 weeks. Pituitary weight increased between 8 and 20 weeks in control rats, whereas it decreased significantly in the irradiated animals. Irradiation induced time- and dose-dependent changes in pituitary hormone contents. GH and PRL were most sensitive and decreased by more than 90% after irradiation; TSH contents were unaffected 8 weeks after the lowest dose of irradiation, but were reduced at 14 and 20 weeks. LH and ACTH were the slowest to be affected, and only at the greater doses of radiation. Thus progressive multiple pituitary endocrine deficits can be induced differentially in rats by increasing doses of cranial irradiation. This model should prove useful for defining the sites and mechanisms by which cranial irradiation induces neuroendocrine dysfunction.

Septo-optic dysplasia (SOD) is a highly variable condition characterized by midline neurological abnormalities associated with pituitary hypoplasia and optic nerve hypoplasia. The aetiology is unknown. Mutant mice, in which a novel homeobox gene, Hesx1, has been disrupted, exhibit a phenotype that resembles the phenotype of SOD. We therefore wished to explore the possibility that this gene is implicated in SOD. We cloned and sequenced the human homologue HESX1 and screened for mutations in affected individuals using single-stranded conformational polymorphism analysis, followed by cloning and sequencing of any exons which showed a band shift. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain, leading to a loss of in vitro DNA binding. Subsequently, we have identified heterozygous mutations in HESX1 that are associated with milder pituitary phenotypes. Our studies indicate a vital role for Hesx1/HESX1 in forebrain and pituitary development in mouse and man, and hence in some cases of SOD.

The sections in this article are: 1 How is the Growth Hormone Secretory Pattern Analyzed? 1.1 General Considerations in Pulse Analysis of Growth Hormone Secretion 1.2 Construction of Data Arrays or Hormone Profiles 1.3 Sampling Interval and Aliasing 1.4 Duration of Sampling 1.5 Discrete or Integrated Samples 1.6 Assays and Noise 1.7 Basic Evaluation 1.8 Stationarization 1.9 Analysis of Profiles 1.10 Time Series Analysis 1.11 Deconvolution Analysis 1.12 Baseline Occupancy 1.13 Chaos 1.14 Neural Networks 2 Characterization of Growth Hormone Secretory Patterns 2.1 Ontogeny of Episodic Growth Hormone Secretion 2.2 Growth Hormone Secretory Patterns in the Prepubertal Period 2.3 Puberty and Effects of Gonadal Steroids on the Growth Hormone Secretory Pattern 2.4 Growth Hormone Secretory Patterns in Adulthood 2.5 Other Endocrine Inputs Affecting Growth Hormone Secretory Patterns 2.6 Extrinsic Factors Affecting Growth Hormone Pulsatility 3 How Does the Growth Hormone Secretory Pattern Reflect the Episodic Nature of Hypothalamic Control Mechanisms? 3.1 Growth Hormone–Releasing Hormone and Somatostatin Interactions Generate Growth Hormone Pulsatility 3.2 Other Growth Hormone Secretogogues 3.3 Growth Hormone Feedback Effects on Endogenous Growth Hormone Pulsatility 4 How is The Growth Hormone Secretory Signal Modified in its Passage from the Pituitary to the Peripheral Target Tissues? 4.1 How Are Growth Hormone Patterns Modified by Interactions with Growth Hormone Binding Proteins? 5 How are the Temporal Elements of the Growth Hormone Signal Detected and Interpreted by the Target Tissues? 5.1 Pattern-Dependent Growth Hormone Responses 5.2 Growth Hormone Patterns and Insulin-Like Growth Factor I Generation 6 What is the Significance of the Growth Hormone Secretory Pattern for Statural Growth? 6.1 Animal Studies 6.2 Human Studies 6.3 Indirect Manipulation of Growth Hormone Secretory Pattern 7 Summary

We have demonstrated previously that pharmacological doses of oestradiol decreased leptin receptor expression in the hypothalamus. We therefore analysed leptin receptor expression during the oestrous cycle in the rat, to establish if acute changes in oestradiol affect leptin receptor expression under physiological conditions. Radioactive in situ hybridization histochemistry was used to measure the gene expression under investigation. Total leptin receptor transcript levels were lowest in pro-oestrus in the choroid plexus, these changes correspond inversely with levels of circulating oestradiol in the rat 4-day oestrous cycle. In contrast full-length leptin receptor levels in both arcuate and ventromedial nuclei did not correspond to the levels of total leptin receptor in the same areas of the hypothalamus or serum levels of oestradiol. Full-length leptin receptor expression in the arcuate nucleus was negatively correlated with neuropeptide Y (NPY) expression (r = 0.447, p < 0.05) in the same nucleus. Arcuate nucleus NPY expression did not correlate significantly with the expression of total leptin receptors in the arcuate nucleus (r = 0.080) or serum leptin levels (r = 0.251). Our results demonstrate that leptin receptor expression is regulated during the oestrous cycle. The unchanged serum leptin levels during the oestrous cycle together with the correlation between the expression of leptin-RL and NPY provide circumstantial evidence that regulation of leptin receptor abundance in the hypothalamus governs the biological actions of leptin.

In addition to stimulating GH release, GH secretagogues such as GH-releasing peptide-6 (GHRP-6) stimulate small amounts of ACTH and PRL release. Although the effects on ACTH have recently been studied, there is little information about the effects of GHRP-6 on PRL. We have now studied GHRP-6-induced GH and PRL release and their regulation by estrogen (E2) in anesthetized male and female rats and in GH-deficient dwarf (dw/dw) rats that maintain high pituitary PRL stores and show elevated hypothalamic GH secretagogue receptor expression. Whereas GHRP-6 (0.1–2.5 μg, iv) did not induce PRL release in normal male or female rats, significant PRL responses were observed in dw/dw females. These responses were abolished by ovariectomy and could be strongly induced in male dw/dw rats by E2 treatment. These effects could be dissociated from GHRP-6-induced GH release in the same animals, but not from PRL release induced by TRH, which was also abolished by ovariectomy and induced in males by E2 treatment. However, the effects of GHRP-6 on PRL were unlikely to be mediated by TRH because in the same animals, TSH levels were unaffected by GHRP-6 whereas they were increased by TRH. The increased PRL response could reflect an increase in GH secretagogue receptor expression that was observed in the arcuate and ventromedial nuclei of E2-treated rats. Our results suggest that the minimal PRL-releasing activity of GHRP-6 in normal rats becomes prominent in GH-deficient female dw/dw rats and is probably exerted directly at the pituitary; these GHRP-6 actions may be modulated by E2 at both hypothalamic and pituitary sites.

Long-acting gonadotropin-releasing hormone (GnRH) analogs (GnRH-a) suppress gonadal steroid production and are used in precocious puberty, resulting in an arrest of pubertal development, a slower epiphyseal maturation, and a deceleration of growth, but an increased final height. However, the way that GnRH-a affect growth is not clear. GnRH-a treatment might not only affect gonadal steroid production but might also modulate the GH axis and thereby affect growth. We used a rat model to investigate the long-term effects of prepubertally started GnRH-a treatment (triptorelin) on growth, spontaneous GH secretion, hepatic GH receptors (GHR), and GH-binding protein (GHBP) and compared it with surgical gonadectomy. Triptorelin affected most parameters in the same direction as surgical gonadectomy but to a lesser extent. In females, growth was enhanced by triptorelin, baseline GH secretion was decreased, and hepatic GHR and GHBP were decreased. Apart from these effects on the GH axis, reduction of the direct inhibiting effect of estrogen on growth could be responsible for the triptorelin-induced growth. In males, triptorelin treatment enhanced body weight gain and slightly enhanced gain in length. GH peak amplitude was the only parameter of GH secretion affected and decreased, whereas GHR or GHBP were not affected. This stimulation of weight gain by long-term triptorelin treatment in male rats, which is opposite the effect of surgical gonadectomy, could indicate an interference of GnRH-a in the hormonal regulation of food intake and body weight control. We conclude that triptorelin treatment affected growth and the GH-GHR-GHBP axis in rats, more markedly in females than in males. However, triptorelin was not as effective as surgical gonadectomy.

International audience; Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hy-pothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome. (Endocrinology 155: 1887-1898, 2014)

In the quest for a better understanding of climate change, greater importance is attached to monitoring the levels of atmospheric carbon dioxide to gain an improved knowledge of sources and sinks. Remote sensing is a critical tool in this research area and differential absorption lidar (DIAL) is one important technique. The laser is the critical component of a DIAL instrument. This paper describes the development of a laser source for the detection and measurement of carbon dioxide. Publisher PDF

Two different dwarf rat models with primary (dw/dw, DW) or secondary (transgenic growth retarded, WF/Tgr) GH deficiency and contrasting hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIH) expression were implanted sc with GC cells. These form encapsulated rat GH-secreting tumors that maintain high plasma rat GH levels for several weeks. In both strains, GC cell tumors stimulated growth and raised GHBP levels, without affecting pituitary GH content. In DW rats, GC cell implants increased SRIH expression in the periventricular nucleus (PeV), but not in the arcuate nucleus (ARC), whereas their high GHRH expression in ARC was decreased by GC cells. In contrast, GC cell implants in WF/Tgr rats had little effect on the already high SRIH expression in PeV or low GHRH expression in ARC, although they reduced SRIH expression in ARC. GC cell implants also reduced GH receptor expression in both ARC and PeV in the WF/Tgr dwarves. Thus, chronic GH overexposure stimulates rapid growth in both dwarf strains, but has differential hypothalamic effects in these models. This experimental approach now makes it possible to study the effects of pathophysiological concentrations of GH ranging from dwarfism to acromegaly in the same animal model.

Synthetic GH secretagogues (GHSs) act via a receptor (GHS-R) distinct from that for GH-releasing hormone (GHRH). We have studied the hypothalamic expression and regulation of this receptor by in situ hybridization using a homologous riboprobe for rat GHS-R. GHS-R mRNA is prominently expressed in arcuate (ARC) and ventromedial nuclei (VMN) and in hippocampus, but not in the periventricular nucleus. Little or no specific hybridization could be observed in the pituitary under the conditions that gave strong signals in the hypothalamus. No sex difference in GHS-R expression was found in ARC or hippocampus, though expression in VMN was lower in males than in females. Compared with GHRH and neuropeptide Y (NPY), GHS-R was expressed in a distinct region of ventral ARC, and in regions of VMN not expressing GHRH or NPY. GHS-R expression was highly sensitive to GH, being markedly increased in GH-deficient dw/dw dwarf rats, and decreased in dw/dw rats treated with bovine GH (200 μg/day) for 6 days. Similar changes w...

To overcome the difficulties of studying hormone pulsatility in the newborn, we have developed an automated microsampling system that permits the measurement of hormones in small prediluted samples of blood (40 microL) taken at 10-min intervals over 12 h. The system has been validated in adult volunteers, and the error attributable to the dilution was

GH-releasing hexapeptide (GHRP-6) is a synthetic secretagogue that stimulates the release of GH by acting at both hypothalamic and pituitary sites. GHRPs also consistently elicit small, but significant, increases in plasma concentrations of ACTH and adrenal steroids. As these secretagogues do not release ACTH directly, they probably interact with the hypothalamic peptidergic systems controlling ACTH release, such as CRH and arginine vasopressin (AVP). We have now examined the activation of the hypothalamo-pituitary-adrenal axis by GHRP-6 in conscious rats. In a series of experiments, rats were injected i.v. with 10 microg GHRP-6, 2 microg CRH, 0.5 microg AVP, or saline, alone or in combination, and serial plasma samples withdrawn and assayed for ACTH, corticosterone, and GH. CRH and AVP increased plasma ACTH levels in all rats, whereas ACTH and corticosterone responses to GHRP-6 were variable and were dependent on the prevailing activity of the hypothalamo-pituitary-adrenal axis. GHRP-6 stimulated the largest ACTH responses in rats that had the lowest basal plasma ACTH and corticosterone levels before GHRP-6 administration. GHRP-6 given in combination with CRH did not increase ACTH levels beyond the response to CRH alone (change in ACTH, 1570 +/- 207 vs. 1714 +/- 245 pg/ml), whereas the combination of GHRP-6 and AVP markedly increased ACTH levels compared with the effects of AVP alone (change in ACTH, 5587 +/- 669 vs. 2338 +/- 451 pg/ml; P0.05). The GH responses to GHRP-6 were significantly greater in rats with low basal plasma ACTH and corticosterone levels than in rats with elevated ACTH and corticosterone levels (change in GH response, 119 +/- 27 vs. 29 +/- 7 ng/ml; P0.01). CRH alone significantly inhibited GH release (pre- vs. 40 min post-CRH, 11.9 +/- 3.8 vs. 1.7 +/- 0.4 ng/ml; P0.05), whereas AVP alone had no effect on GH levels. Neither CRH nor AVP had any effect on the GH response to GHRP-6. We suggest that GHRP-6 acts via the hypothalamus to mediate the release of ACTH, and that these effects are probably mediated at least in part via the release of endogenous CRH and are subject to regulation by circulating glucocorticoids.

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.

We describe a new animal model of obesity and GH deficiency and report the effects on body fat of administering (GH) and insulin-like growth factor (IGF-I) in the model. Female GH-deficient dwarf rats fed a high-fat diet became obese and insulin-resistant compared with chow-fed controls. They were treated with recombinant human GH (rhGH 100-500 micrograms/day, s.c. for 14 days) by daily injection or minipump infusion with or without rhIGF-I (200 micrograms/day, sc infusion). Injections of rhGH increased body weight; infusions of rhGH caused weight loss. RhIGF-I by itself, or rhIGF-I plus GH injections had little effect, whereas rhGH infusions plus rhIGF-I caused a weight loss equivalent to the weight gained during the high-fat feeding and a decrease in fat pad weight. For some responses (serum IGF-1 and GHBP), the obese rats were GH resistant. Fat was lost from the internal fat pads when obese rats were returned to a chow diet, and injections of rhGH surprisingly attenuated this loss of fat. In obese dwarf rats, the lipolytic effects of rhGH are dose-regime dependent. By itself IGF-I is not insulin-like, but in the presence of GH it has antiinsulin actions causing a powerful net lipolysis. If GH plus IGF-I have similar effects in humans they may be useful for reducing body fat.

Body growth, GH secretory pattern, hepatic GH receptor (GHR), and plasma GH-binding protein (GHBP) levels are all sexually dimorphic in the rat. Male rats grow faster than females, and in GH-deficient animals, GH therapy is more effective when given in a pulsatile pattern rather than a continuous infusion. This contrasts with GHBP and hepatic GHR levels, which are lower in males than in females and raised by continuous but not pulsatile GH therapy. One possible explanation is that growth is primarily regulated by GH pulses, whereas GHR and GHBP are regulated mostly by the trough levels (which are lower in males than in females). To test this hypothesis directly, GH-deficient dwarf rats were given patterned iv infusions of hGH in which the relative contributions of the peak and trough components of the GH pattern were systematically varied, independently of dose, and their effects on weight and length gain, plasma GHBP, and hepatic GHR binding were measured. We found that the dose-response curves for GH given by pulsatile vs. continuous infusion were significantly nonparallel, and that growth was primarily stimulated by the pulsatile component of a mixed GH infusion pattern; doubling the GH dose by adding a continuous (c) infusion to a series of pulses (p) neither enhanced nor inhibited weight gain (36 micrograms hGH/day pulses (36p) vs. 36 micrograms hGH/day pulses + 36 micrograms hGH/day continuously (36p + 36c):0.9 +/- 0.2 g/day vs. 1.1 +/- 0.2 g/day), whereas doubling the GH dose by adding a pulsatile component significantly enhanced growth (72p:2.1 +/- 0.2 g/day, P < 0.01). Conversely, hepatic GHR and plasma GHBP levels were highly sensitive to the continuous element of the mixed infusion pattern and were totally unaffected by varying the pulsatile component over a wide range of doses. These results strongly suggest that growth and hepatic GHR/plasma GHBP respond differentially to the peak and trough components of the GH secretion pattern in the rat.

Growth hormone-releasing factor (GRF) regulates GH release and somatotrope proliferation via a specific G-protein-coupled receptor. Little is known about the endocrine factors that regulate the expression of the GRF receptor (GRF-R) in the pituitary gland. We have developed a sensitive solution hybridization/RNAse protection assay for GRF-R mRNA in rat pituitary extracts. GRF-R transcripts were readily detectable in similar amounts in normal male and female rats, but were markedly reduced in extracts from age-matched growth hormone (GH)-deficient dwarf (dw) rats of either sex. The reduced GRF-R expression would appear to reflect somatotrope hypoplasia rather than GH deficiency per se since a similar reduction in GRF-R expression was seen in a transgenic model of dominant dwarfism, whereas GRF-R expression was significantly elevated in rats with GH deficiency induced by hypothyroidism. We were unable to demonstrate significant effects on GRF-R expression with infusions of human GH (hGH) or insulin-like growth factor 1, which stimulate growth in dw rats, but dexamethasone treatment induced a significant, time-related increase in GRF-R mRNA levels. We conclude that this assay can usefully quantify pituitary GRF-R expression in normal rats, and its reduction in two different strains of mutant dwarf rats with somatotrope hypoplasia.

In humans, serum growth hormone (GH) concentrations are significantly higher in women than in men, but the neuroendocrine mechanisms that underlie such gender differences are not known. We compared normal episodic GH secretion in males and females in three distinct settings: two human studies employing quite different assay techniques (immunoradiometric assay and a high-sensitivity immunofluorimetric method) and a rat study. To quantify the amount of regularity in data, we utilized approximate entropy (ApEn), a scale- and model-independent statistic. In each study, females exhibited significantly greater statistical irregularity in GH concentration series than their male counterparts (P < 10(-3) for each human study, P < 10(-6) for the rat study), implying that mass and mode of GH secretion are regulated differently in males and females. The regularity comparisons indicated complete gender separation (100% specificity and sensitivity) for the rat study and nearly complete separation for the immunofluorimetric assay study. The consistency and statistical significance of these findings suggest that this gender difference may be broadly based within higher animals and that this may be readily evaluated objectively by analysis of ApEn.

Alcohol dependence is a common, complex and debilitating disorder with genetic and environmental influences. Here we show that alcohol consumption increases following mutations to the γ-aminobutyric acidA receptor (GABAAR) β1 subunit gene (Gabrb1). Using N-ethyl-N-nitrosourea mutagenesis on an alcohol-averse background (F1 BALB/cAnN x C3H/HeH), we develop a mouse model exhibiting strong heritable preference for ethanol resulting from a dominant mutation (L285R) in Gabrb1. The mutation causes spontaneous GABA ion channel opening and increases GABA sensitivity of recombinant GABAARs, coupled to increased tonic currents in the nucleus accumbens, a region long-associated with alcohol reward. Mutant mice work harder to obtain ethanol, and are more sensitive to alcohol intoxication. Another spontaneous mutation (P228H) in Gabrb1 also causes high ethanol consumption accompanied by spontaneous GABA ion channel opening and increased accumbal tonic current. Our results provide a new and important link between GABAAR function and increased alcohol consumption that could underlie some forms of alcohol abuse.