Persistent changes in somatostatin and neuropeptide Y mRNA levels but not in growth hormone-releasing hormone mRNA levels in adult rats after intrauterine growth retardation

A reduction in the availability of oxygen and nutrients across the placenta in the last trimester of pregnancy may lead to intrauterine growth retardation (IUGR) which, in turn, may cause a persistent postnatal growth failure. However, it is unknown whether this persistent growth retardation is centrally mediated through alterations in the components of the growth hormone (GH)-axis. We tested the hypothesis that alterations in the development of the central components of the GH-axis contribute to the persistent growth failure observed after experimentally induced IUGR or early postnatal food restriction (FR) in the rat. Using semi-quantitative in situ hybridization, we compared somatostatin (SS), GH-releasing hormone (GHRH) and neuropeptide Y (NPY) mRNA levels in adult rats experimentally subjected to IUGR or FR. We report that IUGR increased the expression of SS mRNA in the periventricular nucleus (PeN) of adult male and female rats by 128% and 153% respectively, did not alter the expression of GHRH mRNA in the arcuate nucleus (ARC) and decreased the NPY mRNA expression in the ARC by 73% in males and 61% in females, whereas in the FR group no changes in the expression of these mRNAs were observed. These data show that the timing of malnutrition or the presence of the placenta is important for the long-term alterations since the effects only occurred in the prenatally induced growth retardation and not in the early postnatally induced growth retardation group.     

Sexually dimorphic expression of the growth hormone-releasing hormone gene is not mediated by circulating gonadal hormones in the adult rat

The sexual dimorphism characterizing GH secretion in the rat is thought to be related to differences in the hypothalamic synthesis and release of the GH-regulating peptides, GH-releasing hormone (GHRH), and somatostatin. Therefore, the influence of gender and sex steroid hormones on hypothalamic expression of the GHRH gene in adult rats were examined. GHRH messenger RNA (mRNA) levels were measured in individual rat hypothalami by Northern hybridization analysis using a 32P-labeled complementary DNA encoding rat GHRH. Destruction of hypothalamic GHRH neurons by neonatal treatment with monosodium glutamate caused similar 3-fold reductions in the levels of GHRH mRNA in adult male and female animals. In three separate experiments, hypothalamic GHRH mRNA concentrations in male rats were 2- to 3-fold greater than in randomly cycling females (four or five rats per group; P less than 0.01). In spite of the greater abundance of GHRH mRNA abundance in the male rat hypothalamus, circulating gonadal steroids lacked the ability to modulate GHRH gene expression in adult animals, since neither gonadectomy nor pharmacological sex steroid replacement changed GHRH mRNA levels in the hypothalamus of male and female adult rats. Furthermore, GHRH mRNA concentrations in female rats were similar during the proestrus, estrus, and diestrus phase of the estrous cycle. Also, GH inhibited hypothalamic GHRH gene expression in a sex-specific manner. Exposure to high levels of GH secreted by the MtTW15 tumor for 4 weeks reduced GHRH mRNA concentrations 7-fold in male rats (P less than 0.001) but only 2-fold in females (P less than 0.05). These studies demonstrate that GHRH gene expression in the rat hypothalamus is sexually dimorphic. Basal mRNA levels are greater in male rats, and expression in male hypothalami is more sensitive to feedback inhibition by GH than expression in females. There is no evidence for regulation of GHRH mRNA levels by either testosterone or estrogen in adult rats. These gender differences in GHRH gene expression likely contribute to the generation of a sex-specific pattern of GH secretion.     

Estrogen receptor (ER)alpha, but not ERbeta, gene is expressed in growth hormone-releasing hormone neurons of the male rat hypothalamus

GH synthesis and release from pituitary somatotropes is controlled by the opposing actions of the hypothalamic neuropeptides, GH-releasing hormone (GHRH), and somatostatin (SS). There is a striking sex difference in the pattern of GH secretion in rats. Early reports indicate that gonadal steroids have important imprinting effects during the neonatal period. Recently, our laboratory and others have reported that the GH secretory pattern is altered by short-term gonadal steroid treatment in adult rat, suggesting that gonadal steroids are also important determinants of the pattern of GH secretion during adult life. However, the site of action of gonadal steroids in the adult rat hypothalamus is still unknown. In this study, we used in situ hybridization in the adult male rat brain to determine whether GHRH neurons and/or SS neurons coexpress estrogen receptor alpha (ERalpha) and ERss genes. In the medial basal hypothalamus of adult male rat, the ERalpha messenger RNA (mRNA) was located in medial preoptic area (MPA) and arcuate nucleus (ARC), whereas ERss mRNA was detected in MPA, supraoptic nucleus, and paraventricular nucleus. From studies using adjacent sections, the distribution of ERalpha mRNA-containing cells appeared to overlap in part with those of GHRH and SS expressing cells only in the ARC. On the other hand, the distribution of ERss mRNA-containing cells does not appear to overlap with GHRH cells or SS cells. The double label in situ hybridization studies showed that in the ARC, 70% of GHRH neurons contain ERalpha mRNA, whereas less than 5% of SS neurons expressed the ERalpha gene. These results indicated that GHRH neurons are direct target cells for estrogens, and estrogens may act directly on GHRH neurons through ERalpha during adult life to modify GH secretory patterns.     

Acute and chronic galanin administration decreases hypothalamic galanin synthesis in both male and female adult rats: evidence for a long-loop galanin autofeedback

The secretion of growth hormone (GH) in both male and female rats is controlled by two main neuropeptides, GH-releasing hormone (GHRH), which is stimulatory, and somatostatin, which is inhibitory. Recently, it has been shown that galanin (GAL) also stimulates GH secretion, although the underlying mechanism is still unknown. It was the aim of this study to begin to elucidate if and how GAL regulates its own production at the hypothalamic and pituitary level. Rats underwent the following experimental trials. In experiment 1, adult male and female rats had blood samples collected at -15 minutes, -7.5 minutes, and immediately preceding a subcutaneous (s.c.) injection of GAL at a dose of either 50 or 200 microg/kg. Blood samples were collected at 5, 10, 15, 30, and 60 minutes, and the GH concentration was measured using a radioimmunoassay. The tissues were collected and analyzed for mRNA levels of hypothalamic and pituitary GAL. In experiment 2, adult male and female rats were treated long-term with 200 microg/kg GAL for 7 days s.c., and the pituitary and hypothalamus were analyzed for GAL mRNA. Serum GH concentrations were significantly increased in acutely dosed male and female rats regardless of the dosage level. For the male and female animals acutely dosed with both 50 and 200 microg/kg GAL, hypothalamic GAL mRNA was decreased, whereas pituitary GAL mRNA was affected by 200 microg/kg GAL only in females (increased). For the animals treated long-term with GAL, hypothalamic GAL mRNA was decreased while mRNA for pituitary GAL was increased. We conclude that regardless of the dosage and duration of treatment, administration of GAL negatively regulates hypothalamic GAL mRNA in a non-gender-specific way. Pituitary GAL synthesis appears to be stimulated particularly during chronic SCGAL administration.     

Growth hormone-releasing hormone (GHRH) neurons in the arcuate nucleus (Arc) of the hypothalamus are decreased in transgenic rats whose expression of ghrelin receptor is attenuated: Evidence that ghrelin receptor is involved in the up-regulation of GHRH expression in the arc

GH secretagogue (GHS)/ghrelin stimulates GH secretion by binding mainly to its receptor (GHS-R) on GHRH neurons in the arcuate nucleus (Arc) of the hypothalamus. GHRH, somatostatin, and neuropeptide Y (NPY) in the hypothalamus are involved in the regulatory mechanism of GH secretion. We previously created transgenic (Tg) rats whose GHS-R expression is reduced in the Arc, showing lower body weight and shorter nose-tail length. GH secretion is decreased in female Tg rats. To clarify how GHS-R affects GHRH expression in the Arc, we compared the numbers of GHS-R-positive, GHRH, and NPY neurons between Tg and wild-type rats. Immunohistochemical analysis showed that the numbers of GHS-R-positive neurons, GHRH neurons, and GHS-R-positive GHRH neurons were reduced in Tg rats, whereas the numbers of NPY neurons and GHS-R-positive NPY neurons did not differ between the two groups. The numbers of Fos-positive neurons and Fos-positive GHRH neurons in response to KP-102 were decreased in Tg rats. Competitive RT-PCR analysis of GHRH mRNA expression in the cultured hypothalamic neurons showed that KP-102 increased NPY mRNA expression level and that NPY decreased GHRH mRNA expression level. KP-102 increased GHRH mRNA expression level in the presence of anti-NPY IgG. GH increased somatostatin mRNA expression. Furthermore, GH and somatostatin decreased GHRH mRNA expression, whereas KP-102 showed no significant effect on somatostatin mRNA expression. These results suggest that GHS-R is involved in the up-regulation of GHRH and NPY expression and that NPY, somatostatin, and GH suppress GHRH expression. It is also suggested that the reduction of GHRH neurons of Tg rats is induced by a decrease in GHS-R expression.     

Sexual dimorphism of growth hormone-releasing hormone and somatostatin gene expression in the hypothalamus of the rat during development.

The secretory pattern of GH secretion is markedly sexually dimorphic in the adult rat. The patterning of GH secretion is determined by the coordinated activity of somatostatin (SS)- and GH-releasing hormone (GHRH)-containing neurosecretory cells located in the hypothalamus. In this study we examined whether there is sexual dimorphism in the expression of the SS and GHRH genes and, if so, at what developmental stage this becomes evident. To address these questions, we measured SS messenger RNA (mRNA) levels in neurons of the periventricular nucleus and GHRH mRNA levels in the arcuate nucleus and ventromedial nucleus of the hypothalamus in male and female rats at 10, 25, 35, and 75 days of age. Using in situ hybridization and a computerized image analysis system, we measured SS mRNA and GHRH mRNA signal levels in individual neurons and compared these levels among the different age groups. We found that male animals had significantly higher levels of SS mRNA than females at every age. Similarly, males had higher GHRH mRNA levels than females; however, this difference was statistically significant only at 10 and 75 days of age. Developmental changes in GHRH mRNA levels were similar for both sexes, with GHRH message levels increasing gradually over the course of maturation. SS mRNA signal levels also changed over the course of development in both male and female animals. In the male rat, SS mRNA levels increased significantly between 10 and 25 days of age and declined significantly between 35 and 75 days of age. In the female rat, SS mRNA levels increased gradually between 10 and 35 days of age, then, as in the male, declined significantly between days 35 and 75. We conclude that sex differences and age-dependent changes in the expression of the SS and GHRH genes may subserve the sexual dimorphism and developmental alterations in the pattern of GH secretion in the rat.     

Expression analysis of hypothalamic and pituitary components of the growth hormone axis in fasted and streptozotocin-treated neuropeptide Y (NPY)-intact (NPY+/+) and NPY-knockout (NPY-/-) mice

In the fasted and the streptozotocin (STZ)-induced diabetic male rat, hypothalamic growth hormone (GH)-releasing hormone (GHRH) mRNA levels, and pulsatile GH release are decreased. These changes are believed to be due to a rise in hypothalamic neuropeptide Y (NPY) that inhibits GHRH expression. To directly test if NPY is required for metabolic regulation of hypothalamic neuropeptides important in GH secretion, NPY, GHRH and somatostatin (SRIH) mRNA levels were determined in fasted (48 h) and STZ-treated wild-type (NPY(+/+)) and NPY-knockout (NPY(-/-)) mice by ribonuclease protection assay. In addition, pituitary receptor mRNA levels for GHRH (GHRH-R), ghrelin (GHS-R) and SRIH (sst2) were assessed by RT-PCR. Under fed conditions the GH axis of NPY(+/+) and NPY(-/-) did not differ. In the NPY(+/+) mouse, fasting resulted in a 23% weight loss and >250% increase in NPY mRNA accompanied by a significant reduction in both GHRH and SRIH mRNA. These changes were associated with increases in pituitary expression of GHRH-R and GHS-R and a concomitant suppression of sst2. In the NPY(-/-) mouse, fasting also resulted in a 23% weight loss and comparable changes in GHRH-R and sst2, but failed to alter GHRH, SRIH and GHS-R mRNA levels. Fasting resulted in an overall increase in circulating GH, which reached significance in the fasted NPY(-/-) mouse. Induction of diabetes in NPY(+/+) mice, using a single, high-dose, STZ injection (150 mg/kg), resulted in modest weight loss (5%), and a 158% increase NPY expression which was associated with reciprocal changes in pituitary GHS-R and sst2 expression, similar to that observed in the fasted state, but no change in hypothalamic GHRH or SRIF expression was observed. Induction of diabetes in NPY(+/+) and NPY(-/-) mice, using a multiple, low-dose, STZ paradigm (5 consecutive daily injections of 40 mg/kg), did not alter body weight, hypothalamic neuropeptide expression or pituitary receptor expression, with the exception that sst2 mRNA levels were suppressed and GH levels did rise in the NPY(-/-) mouse. These observations demonstrate that NPY is not required for basal regulation of the GH axis, but is required for fasting-induced suppression of GHRH and SRIH expression, as well as fasting-induced augmentation of pituitary GHS-R mRNA. In contrast to the rat, fasting clearly did not suppress circulating GH levels in mice, but resulted in an overall rise in mean GH levels, similar to that observed in other mammalian species. The fact that many of the fasting-induced changes in the GH axis were observed in the high-dose STZ-treated mice, but were not observed in the multiple, low-dose paradigm, suggests STZ-mediated modulation of GH axis function is dependent on the severity of the catabolic state and not hyperglycemia.     

Maternal perinatal undernutrition drastically reduces postnatal leptin surge and affects the development of arcuate nucleus proopiomelanocortin neurons in neonatal male rat pups

A growing body of evidence suggests that maternal undernutrition sensitizes the offspring to the development of energy balance metabolic disorders such as type 2 diabetes, dyslipidemia, and obesity. The present study aimed at examining the impact of maternal undernutrition on leptin plasma levels in newborn male rats and on the arcuate nucleus proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons that are major leptin targets. Using a model of perinatal maternal 50% food-restricted diet (FR50) in the rat, we evaluated leptin plasma levels and hypothalamic POMC and NPY gene expression from postnatal day (PND) 4 to PND30 in both control and FR50 offspring. In control rats, a postnatal peak of plasma leptin was observed between PND4 and PND14 that reached a maximal value at PND10 (5.17 +/- 0.53 ng/ml), whereas it was dramatically reduced in FR50 pups with the higher concentration at PND7 (0.93 +/- 0.23 ng/ml). In FR50 animals, using semiquantitative RT-PCR and in situ hybridization, we showed that the hypothalamic POMC mRNA level was decreased from PND14 until PND30, whereas NPY gene expression was not significantly modified. In PND21 FR50 animals, we observed strikingly reduced immunoreactive beta-endorphin nerve fibers projecting to the hypothalamic paraventricular nucleus without affecting NPY projections. Our data showed that maternal undernutrition drastically reduces the postnatal surge of plasma leptin, disturbing particularly the hypothalamic wiring as well as the gene expression of the anorexigenic POMC neurons in male rat pups. These alterations might contribute to the adult metabolic disorders resulting from perinatal growth retardation.     

Differential responses of the growth hormone axis in two rat models of streptozotocin-induced insulinopenic diabetes

The impact of streptozotocin (STZ)-induced, insulinopenic diabetes on the GH axis of rats and mice differs from study to study, where this variation may be related to the induction scheme, severity of the diabetes and/or the genetic background of the animal model used. In order to begin differentiate between these possibilities, we compared the effects of two different STZ induction schemes on the GH axis of male Sprague-Dawley rats: (1) a single high-dose injection of STZ (HI STZ, 80 mg/kg, i.p.), which results in rapid chemical destruction of the pancreatic beta-cells, and (2) multiple low-dose injections of STZ (LO STZ, 20 mg/kg for 5 consecutive days, i.p.), which results in a gradual, autoimmune destruction of beta-cells. STZ-treated animals were killed after 3 weeks of hyperglycemia (>400 mg/dl), and in both paradigms circulating insulin levels were reduced to <40% of vehicle-treated controls. HI STZ-treated rats lost weight, while body weights of LO STZ-treated animals gradually increased over time, similar to vehicle-treated controls. As previously reported, HI STZ resulted in a decrease in circulating GH and IGF-I levels which was associated with a rise in hypothalamic neuropeptide Y (NPY) mRNA (355% of vehicle-treated controls) and a fall in GH-releasing hormone (GHRH) mRNA (45% of vehicle-treated controls) levels. Changes in hypothalamic neuropeptide expression were reflected by an increase in immunoreactive NPY within the arcuate and paraventricular nuclei and a decrease in GHRH immunoreactivity in the arcuate nucleus, as assessed by immunohistochemistry. Consistent with the decline in circulating GH and hypothalamic GHRH, pituitary GH mRNA levels of HI STZ-treated rats were 58% of controls. However, pituitary receptor mRNA levels for GHRH and ghrelin increased and those for somatostatin (sst2, sst3 and sst5) decreased following HI STZ treatment. The impact of LO STZ treatment on the GH axis differed from that observed following HI STZ treatment, despite comparable changes in circulating glucose and insulin. Specifically, LO STZ treatment did suppress circulating IGF-I levels to the same extent as HI STZ treatment; however, the impact on hypothalamic NPY mRNA levels was less dramatic (158% of vehicle-treated controls) where NPY immunoreactivity was increased only within the paraventricular nucleus. Also, there were no changes in circulating GH, hypothalamic GHRH or pituitary receptor expression following LO STZ treatment, with the exception that pituitary sst3 mRNA levels were suppressed compared with vehicle-treated controls. Taken together these results clearly demonstrate that insulinopenia, hyperglycemia and reduced circulating IGF-I levels are not the primary mediators of hypothalamic and pituitary changes in the GH axis of rats following HI STZ treatment. Changes in the GH axis of HI STZ-treated rats were accompanied by weight loss, and these changes are strikingly similar to those observed in the fasted rat, which suggests that factors associated with the catabolic state are critical in modifying the GH axis following STZ-induced diabetes.     

Modulation of growth axis gene expression by in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the weaning Holtzman rat

While thein utero and lactational effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on both male and female reproductive systems appear to be severe, little is known about its effects on the developing growth axis. The objective of this study was to describe changes in growth axis gene expression that accompany exposure to TCDD duringin utero and lactational development. Pregnant Holtzman rats were administered 1 μg TCDD/kg maternal body weight or vehicle control on gestational day 15 by gavage. Using ribonuclease protection assays, we compared mRNA levels measured in 21-d-old female pups exposed to TCDD with levels measured in control animals for the following genes: somatostatin, growth hormone-releasing hormone (GHRH), hypothalamic and pituitary galanin (GAL), growth hormone (GH), and insulin-like growth factor-I (IGF-I). Serum GH concentrations measured by radio-immunoassay were significantly increased, although GH mRNA levels were unchanged from controls by TCDD exposure. Hypothalamic GAL mRNA was decreased in TCDD-treated animals, whereas pituitary GAL mRNA in TCDD-treated animals was not altered. GHRH mRNA was increased in hypothalami from TCDD-exposed animals. IGF-I mRNA in the liver was decreased to 67% of controls. These data indicate that the growth axis is sensitive to the effects of TCDD delivered during critical periods of development. The alterations observed in growth axis gene expression with exposure to TCDD add to the body of data demonstrating a potent effect of this compound on the fetal and neonatal endocrine system.     

Growth hormone receptor messenger ribonucleic acid distribution in the adult male rat brain and its colocalization in hypothalamic somatostatin neurons.

The activity of both somatostatin (SS) and GH-releasing hormone (GHRH) neurons within several hypothalamic nuclei is regulated, in part, by the feedback effects of GH. However, whether GH, or its intermediate, insulin-like growth factor I, acts on these neurons to alter the synthesis and release of SS and GHRH is unknown. We argued that if GH itself acts directly on the brain to govern its own secretion, then regions of the brain containing SS and GHRH neurons may express the GH receptor gene. We tested this hypothesis by performing in situ hybridization for GH receptor messenger RNA (mRNA) and mapping its distribution in the brain. We observed GH receptor mRNA-containing cells in various brain regions including the thalamus, septal region, hippocampus, dentate gyrus, amygdala, and hypothalamus. Next we sought evidence for expression of the GH receptor mRNA by SS neurons in the hypothalamus. We addressed this by performing a double-label in situ hybridization to identify neurons expressing both SS mRNA and GH receptor mRNA. We report that SS neurons in the periventricular nucleus and in the paraventricular nucleus coexpress the GH receptor gene, whereas few, if any, of the SS neurons in the cortex express detectable amounts of the GH receptor mRNA. These findings suggest that GH acts directly on the brain and participates in the regulation of its own secretion through a direct action on hypothalamic SS neurons.     

Effects of galanin on growth hormone and prolactin secretion in anorexia nervosa

Galanin (GAL) elicits growth hormone (GH) release in normal subjects through interaction with hypothalamic somatostatin. GAL also stimulates GH-releasing hormone (GHRH) secretion in vitro. In rats, GAL is able to stimulate prolactin (PRL) release, but this effect is not clear in humans. We have thus investigated GAL effects on GH and PRL release in patients with anorexia nervosa (AN), known to have altered regulation of the GH-insulin-like growth factor axis and PRL dynamics, and compared the effects of GHRH and GAL on GH and PRL secretion in AN and normal healthy subjects. Eight women with AN (15 to 27 years; body mass index [BMI], 17 to 19.5 kg/m2) were treated with (1) GHRH 50 microg intravenous (IV) injection, (2) porcine GAL 500 microg infusion from -10 to +30 minutes, and (3) 135-minutes saline infusion as a control, respectively. Both peptides induced a significant increase in plasma GH in AN patients (peak level, 27.41 +/- 5.50 microg/L after GAL and 18.97 +/- 2.67 microg/L after GHRH). When data for AN patients and the control group were compared, GH peak levels after GAL were significantly higher in AN patients (27.41 +/- 5.50 v 13.64 +/- 2.32 microg/L), while GH peak levels after GHRH were not different between the 2 groups (18.97 +/- 2.67 v 15.98 +/- 3.88 microg/L). PRL levels significantly increased after both GHRH (peak, 11.70 +/- 2.80 microg/L) and GAL (peak, 18.02 +/- 5.10 microg/L) treatment in AN patients, but not in normal subjects. We conclude that GAL stimulates exaggerated GH release in AN patients as compared with normal controls, suggesting a dual hypothalamic interaction via both an increase in endogenous GHRH and a decrease in somatostatin secretion. Finally, GAL may act as a PRL secretagogue in AN patients.     

Chronic food-restriction alters the expression of somatostatin and growth hormone-releasing hormone in the ovariectomised ewe.

Changes in the secretion of GH induced by long-term alterations in nutritional status are thought to result from alterations in somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) at the level of the hypothalamus. To date however, the effect of nutrition on the gene expression of SRIF and GHRH in a species where GH secretion is increased by food restriction, as is the case for the sheep and human, remains unknown. We determined the effect of under-nutrition on the expression of SRIF and GHRH in the hypothalamus of sheep. Ovariectomised ewes were randomly divided into two groups and either fed an ad lib diet (n=6) or a restricted diet of 500 g lucerne chaff per day (food-restricted; n=5) for 7 months. In situ hybridisation was used to study hypothalamic gene expression for GHRH, SRIF and galanin (GAL). The food-restricted animals had elevated plasma concentrations of GH; this was associated with an increase in GHRH mRNA levels in the arcuate nucleus (ARC) and reduced SRIF in the rostral periventricular nucleus and ventromedial hypothalamic nucleus. The level of gene expression of GAL in the ARC and SRIF in the caudal periventricular nucleus was similar in ad lib and food-restricted animals. In conclusion, the effect of chronic food-restriction on the secretion of GH reflects increased GHRH and reduced SRIF synthesis in the hypothalamus.     

Endocrine regulation and extended follow up of longitudinal growth in intrauterine growth-retarded rats

Bilateral uterine artery ligation in late gestation was performed in pregnant dams in order to determine the effects of intrauterine growth retardation (IUGR) on long-term postnatal somatic growth and the GH neuroendocrine axis in the adult female and male rat. Body weight (BW), nose-anus length (NAL) and tail length (TL) were recorded at regular intervals in both the IUGR and control (CON) offspring until the age of 93 days. Spontaneous 6-h GH secretory profiles and serum IGF-I were determined around the age of 100 days in both the IUGR and the CON group. No catch-up growth in BW, NAL or TL was observed in young adult male IUGR rats. Female IUGR rats did catch up in NAL beyond the age of 57 days and in TL before weaning, but did not catch up at any time in BW. Spontaneous 6-h GH secretory profiles in female and male IUGR rats at a mean age of 100+/-4 days were similar to their controls at a mean age of 101+/-4 days. Overall median 6-h rat GH plasma concentrations, rat GH peak amplitude, number of rat GH peaks and sum of peak area were not significantly different. Median serum IGF-I levels in young adult female and male IUGR rats showed no difference when compared with their respective controls. These results demonstrate that IUGR, after bilateral uterine artery ligation in late gestation, leads to incomplete BW catch-up growth in young adult rats of both sexes with physiological GH/IGF-I secretion, suggesting intrauterine modulation of tissue responsiveness to GH and IGF-I. Female IUGR rats do catch up in NAL and TL, developing disturbed body proportions.     

The growth hormone (GH)-axis of GH receptor/binding protein gene-disrupted and metallothionein-human GH-releasing hormone transgenic mice: hypothalamic neuropeptide and pituitary receptor expression in the absence and presence of GH feedback

Elevation of circulating GH acts to feed back at the level of the hypothalamus to decrease GH-releasing hormone (GHRH) and increase somatostatin (SRIF) production. In the rat, GH-induced changes in GHRH and SRIF expression are associated with changes in pituitary GHRH receptor (GHRH-R), GH secretagogue receptor (GHS-R), and SRIF receptor subtype messenger RNA (mRNA) levels. These observations suggest that GH regulates its own synthesis and release not only by altering expression of key hypothalamic neuropeptides but also by modulating the sensitivity of the pituitary to hypothalamic input, by regulating pituitary receptor synthesis. To further explore this possibility, we examined the relationship between the expression of hypothalamic neuropeptides [GHRH, SRIF, and neuropeptide Y (NPY)] and pituitary receptors [GHRH-R, GHS-R, and SRIF receptor subtypes (sst2 and sst5)] in two mouse strains with alterations in the GH-axis; the GH receptor/binding protein gene-disrupted mouse (GHR/BP-/-) and the metallothionein promoter driven human GHRH (MT-hGHRH) transgenic mouse. In GHR/BP-/- mice, serum insulin-like growth factor I levels are low, and circulating GH is elevated because of the lack of GH negative feedback. Hypothalamic GHRH mRNA levels in GHR/BP-/- mice were 232 +/- 20% of GHR/BP+/+ littermates (P < 0.01), whereas SRIF and NPY mRNA levels were reduced to 86 +/- 2% and 52 +/- 3% of controls, respectively (P < 0.05; ribonuclease protection assay). Pituitary GHRH-R and GHS-R mRNA levels of GHR/BP-/- mice were elevated to 275 +/- 55% and 319 +/- 68% of GHR/BP+/+ values (P < 0.05, respectively), whereas the sst2 and sst5 mRNA levels did not differ from GHR/BP intact controls as determined by multiplex RT-PCR. Therefore, in the absence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor stimulation of GH synthesis and release. In MT-hGHRH mice, ectopic hGHRH transgene expression elevates circulating GH and insulin-like growth factor I. In this model of GH excess, endogenous (mouse) hypothalamic GHRH mRNA levels were reduced to 69 +/- 6% of nontransgenic controls, whereas SRIF mRNA levels were increased to 128 +/- 6% (P < 0.01). NPY mRNA levels were not significantly affected by hGHRH transgene expression. Also, MT-hGHRH pituitary GHRH-R and GHS-R mRNA levels did not differ from controls. However, sst2 and sst5 mRNA levels in MT-hGHRH mice were increased to 147 +/- 18% and 143 +/- 16% of normal values, respectively (P < 0.05). Therefore, in the presence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor suppression of GH synthesis and release.     

The regulation of GH secretion by sex steroids

Gonadal sex steroids modulate GH synthesis and secretion with effects on both the hypothalamus and anterior pituitary. In the post-pubertal animal, androgens and oestrogens modulate hypothalamic somatostatin (SS) and GHRH synthesis respectively. These effects may be direct as SS neurons express the androgen receptor and many GHRH neurons are oestrogen receptor positive. The neonatal steroid environment modulates the number of GHRH neurons in the adult hypothalamus, as well as their responsivity to post-pubertal steroids. Furthermore, both neonatal and post-pubertal steroids modulate hypothalamic synaptic organisation affecting the number of synaptic inputs and the morphology of glial cells. This in turn has important effects on the ability of the hypothalamus to drive the secretory pulsatility of anterior pituitary hormone release. At the level of the somatotroph, androgens and oestrogens have been reported to stimulate, inhibit or have no effect on GH synthesis. In primary cultures, we found no effect of either androgens or oestrogens on GH mRNA levels. However, the sex steroid environment significantly modified the response of somatotrophs to SS. Furthermore, males have more somatotrophs compared with female rats and this partially depends on the neonatal sex steroid environment. In conclusion, sex steroids have both organisational and activational effects on the GH axis. These effects range from modulating the number of hypothalamic neurons controlling GH secretion, their responsiveness to later steroids, and the synaptic connectivity and neuropeptide production, to modulation of somatotroph numbers in the anterior pituitary and their responsiveness to inputs controlling GH synthesis and secretion.     

Inhibitory effects of ethanol on the growth hormone (GH)-releasing hormone-GH-insulin-like growth factor-I axis in the rat.

Ethanol administration decreases GH secretion in humans and experimental animals. The mechanism of these inhibitory effects was investigated by evaluating the spontaneous secretory pattern of GH in chronically cannulated unanesthetized rats, plasma insulin-like growth factor-I (IGF-I) concentrations, and hypothalamic GH-releasing hormone (GHRH) and somatostatin, and pituitary GH mRNA levels. Body weight gain was reduced in ethanol (5%)-liquid diet-fed rats (n = 6) for 6 days compared to that in both isocalorically pair-fed controls (n = 6) and ad libitum-fed animals (n = 6). Spontaneous GH secretion was markedly decreased (by 75-90%) in ethanol-fed rats compared to that in pair-fed and ad libitum-fed groups, while pulsatile pattern of GH release was preserved, with secretory bursts occurring every 180-220 min in all groups. Mean 6-h plasma GH levels in ethanol-, pair-, and ad libitum-fed animals were: 18.8 +/- 4.5, 113.3 +/- 14.9, and 179.6 +/- 30.1 ng/ml, respectively (P < 0.01, ethanol vs. each control). Plasma IGF-I concentrations were decreased in the ethanol-fed rats (338 +/- 16 ng/ml) compared to those in pair-fed (427 +/- 39 ng/ml; P < 0.05) and ad libitum-fed (769 +/- 25 ng/ml; P < 0.01) rats. Ethanol treatment decreased GHRH mRNA levels to 9% of those in ad libitum-fed (P < 0.01) and 20% of those in pair-fed (P < 0.05) animals, whereas it did not significantly alter somatostatin or GH mRNA levels. The results indicate that the effects of ethanol inhibit GH secretion primarily at the hypothalamic level, resulting in impaired GHRH gene expression. Since the GHRH-GH-IGF-I axis has an important role in growth regulation, the growth retardation seen in experimental models of alcohol abuse may be a consequence at least in part of the suppressive effects of ethanol on this axis.