Hypothalamic SRIF Research Articles

Somatostatin Receptors Type 2 and 5 Expression and Localization During Human Pituitary Development

Somatostatin (SRIF), by acting mainly through sst2 and sst5 receptors, is a potent inhibitor of hormonal secretion by the human anterior pituitary gland. However, the pattern of protein expression of these SRIF receptors remains unknown during pituitary development. To get further insights into the physiological role of SRIF receptors in human development and pituitary function, the present study examined the developmental expression of the sst2 and sst5 receptors in the individual cell types of the anterior human pituitary. Thirteen fetal human pituitaries were investigated between 13 to 38 weeks of gestation (WG) by double-labeling immunofluorescence with antibodies raised against sst2 or sst5 receptors and GH, LH, FSH, TSH, or pro-opiomelanocortin proteins. SRIF immunoreactivity in the hypothalamus and median eminence was investigated at the same developmental ages. Immunoreactivity for the sst2 receptor was evident as early as 13 to 15 WG and onward mainly in TSH-, LH-, and FSH-expressing cells, whereas sst5 immunoreactivity was apparent at the late development stages (35-38 WG). GH-expressing cells mainly expressed sst5 immunoreactivity. SRIF-positive fibers and cells were detected as soon as 13 to 16 WG in the hypothalamus and median eminence and their densities increased with gestational age. The early appearance of hypothalamic SRIF cells and fibers suggests a physiological link between SRIF and its receptors during pituitary development. Whereas sst2 receptors might play a primary role in the differentiation and regulation of TSH, LH, and FSH cells, sst5 receptors appear to be mainly involved in GH regulation from birth onward.

Reduced Somatostatin in Hypothalamus of Young Male Mouse Increases Local but not Circulatory GH

The release of growth hormone (GH) from the pituitary gland is primarily inhibited by somatostatin (SRIF) from the hypothalamus via interactions with five types of SRIF receptors (SSTRs). However, the inhibition mechanism of SRIF on GH has not been fully examined. In this study, we repressed the hypothalamic SRIF in young male mice by stereotaxic injection of the lentiviral-shRNA against SRIF to investigate the role of hypothalamic SRIF on hormone secretion in the GH/IGF-1 axis. We found that the reduction of SRIF in hypothalamus was associated with an increase in the protein, but not the mRNA level, of the GH in the pituitary where SSTR 2 and SSTR 5 act importantly. Interestingly, the level of blood circulatory SRIF, GH, IGF-1 and the body weight were not significantly influenced by the downregulation of hypothalamic SRIF. Our findings provide insights into the mechanisms underlying the inhibition of SRIF on GH secretion.

Constitutive Somatostatin Receptor Activity Determines Tonic Pituitary Cell Response

Somatostatin (SRIF) binds G protein-coupled SRIF receptor subtypes (SST1, -2, -3, -4, and -5) to regulate cell secretion and proliferation. Hypothalamic SRIF inhibits pituitary growth hormone, thyroid stimulating hormone, and ACTH secretion. We tested SRIF-independent constitutive SST activity in AtT20 mouse pituitary corticotroph cells in which ACTH secretion is highly sensitive to SRIF action. Stable transfectants expressing SST2 or SST5 were sensitized to selective agonist action, and constitutive SST receptor activity was demonstrated by forskolin and pertussis toxin cAMP cell responses. Persistent constitutive SST activity decreased cell ACTH responses to CRH through decreased expression of CRH receptor subtype 1. Decreased dopamine receptor type 1 expression was associated with attenuated dopamine agonist action, whereas responses to isoproterenol were enhanced through increased β2-adrenoreceptor expression. Thus, integrated pituitary cell ACTH regulation is determined both by phasic SRIF action, as well as by tonic constitutive SST activity, independently of SRIF.

Constitutive Somatostatin Receptor Activity Determines Tonic Pituitary Cell Response

Somatostatin (SRIF) binds G protein-coupled SRIF receptor subtypes (SST1, -2, -3, -4, and -5) to regulate cell secretion and proliferation. Hypothalamic SRIF inhibits pituitary growth hormone, thyroid stimulating hormone, and ACTH secretion. We tested SRIF-independent constitutive SST activity in AtT20 mouse pituitary corticotroph cells in which ACTH secretion is highly sensitive to SRIF action. Stable transfectants expressing SST2 or SST5 were sensitized to selective agonist action, and constitutive SST receptor activity was demonstrated by forskolin and pertussis toxin cAMP cell responses. Persistent constitutive SST activity decreased cell ACTH responses to CRH through decreased expression of CRH receptor subtype 1. Decreased dopamine receptor type 1 expression was associated with attenuated dopamine agonist action, whereas responses to isoproterenol were enhanced through increased beta2-adrenoreceptor expression. Thus, integrated pituitary cell ACTH regulation is determined both by phasic SRIF action, as well as by tonic constitutive SST activity, independently of SRIF.

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.

Interactions of Growth Hormone Secretagogues and Growth Hormone-Releasing Hormone/Somatostatin

The class of novel synthetic compounds termed growth hormone secretagogues (GHSs) act in the hypothalamus through, as yet, unknown pathways. We performed physiologic and histochemical studies to further understand how the GHS system interacts with the well-established somatostatin (SRIF)/growth hormone-releasing hormone (GHRH) neuroendocrine system for regulating pulsatile GH secretion. Comparison of the GH-releasing activities of the hexapeptide growth hormone-releasing peptide-6 (GHRP-6) and GHRH administered intravenously to conscious adult male rats showed that the pattern of GH responsiveness to GHRP-6 was markedly time-dependent, similar to that observed with GHRH. Immunoneutralization of endogenous SRIF reversed the blunted GH response to GHRP-6 at trough times, suggesting that GHRP-6 neither disrupts nor inhibits the cyclical release of endogenous hypothalamic SRIF. By striking contrast, passive immunization with anti-GHRH serum virtually obliterated the GH responses to GHRP-6, irrespective of the time of administration. These findings suggest that the GHSs do not act by altering SRIF release but, rather, stimulate GH release via GHRH-dependent pathways. Our dual chromogenic and autoradiographic in situ hybridization experiments revealed that a subpopulation of GHRH mRNA-containing neurons in the arcuate (Arc) nucleus and ventromedial nucleus (VMN) of the hypothalamus expressed the GHS receptor (GHS-R) gene. These results provide strong anatomic evidence that GHSs may directly stimulate GHRH release into hypophyseal portal blood, and thereby influence GH secretion, through interaction with the GHS-R on GHRH- containing neurons. Altogether, these findings support the notion that an additional neuroendocrine pathway may exist to regulate pulsatile GH secretion, possibly through the influence of the newly discovered GHS natural peptide, ghrelin.

Differential in vivo regulation of the pituitary growth hormone-releasing hormone (GHRH) receptor by GHRH in young and aged rats.

In aging, alterations of pituitary GH-releasing hormone (GHRH) receptor (GHRH-R)-binding sites have been proposed as one of the initiating factors contributing to the loss of somatotroph responsiveness to GHRH. Changes in the characteristics and/or concentration of the functional GHRH-R could take place in the course of aging and reduce the sensitivity of the somatotroph axis to GHRH. Because chronic exposure to GHRH has been proposed to resensitize aged somatotroph cells, better knowledge of its effects on the regulation of the somatotroph axis is required, particularly at the level of GHRH-R. Two- and 18-month-old male Sprague Dawley rats were treated for 14 days with a daily s.c. injection of 0.5 or 1.0 mg/kg BW human GHRH-(1-29)NH2 or saline. In 2-month-old rats, treatment with 0.5 mg/kg GHRH increased the number of high affinity pituitary GHRH-R-binding sites by 2-fold (P < 0.05) and hypothalamic somatostatin (SRIF) content by 45% (P < 0.05). It did not affect hypothalamic GHRH content, serum total insulin-like growth factor I (IGF-I), or body weight gain. Treatment with 1.0 mg/kg GHRH decreased the number of high affinity pituitary GHRH-R-binding sites by 2.4-fold compared with that in rats treated with 0.5 mg/kg BW (P < 0.05) and increased hypothalamic SRIF content by 45% (P < 0.05), but did not affect GHRH content. It also decreased circulating levels of IGF-I by 13% (P < 0.05) and slowed the growth rate by 17% (P < 0.05). In 18-month-old rats, treatment with 0.5 mg/kg GHRH for 14 days was not sufficient to rejuvenate pituitary GHRH binding parameters. However, treatment with 1.0 mg/kg GHRH restored the affinities of high and low affinity classes of GHRH-binding sites to values similar to those found in 2-month-old rats. Binding capacities of the high and low affinity classes of sites were increased by 1.8- and 3-fold, respectively, although significance was only reached for the low affinity site (P < 0.05). These changes were associated with a normalization of the level of 2.5-kb GHRH-R messenger RNA transcript, which was decreased by 31% in aging rats (P < 0.05), and by a trend for an increase in the 4-kb GHRH-R messenger RNA transcript, which was already increased by 49% in 18-month-old rats (P < 0.05). A normalization of serum IGF-I levels, which were decreased by 11% in 18-month-old control rats (P < 0.01), was also observed. No treatment effect was detected on body weight or hypothalamic SRIF and GHRH contents. We conclude that a 14-day administration of GHRH induces a differential GHRH-R-mediated regulation at the level of the pituitary and probably the hypothalamus as a function of age.

Differential in Vivo Regulation of the Pituitary Growth Hormone-Releasing Hormone (GHRH) Receptor by GHRH in Young and Aged Rats

In aging, alterations of pituitary GH-releasing hormone (GHRH) receptor (GHRH-R)-binding sites have been proposed as one of the initiating factors contributing to the loss of somatotroph responsiveness to GHRH. Changes in the characteristics and/or concentration of the functional GHRH-R could take place in the course of aging and reduce the sensitivity of the somatotroph axis to GHRH. Because chronic exposure to GHRH has been proposed to resensitize aged somatotroph cells, better knowledge of its effects on the regulation of the somatotroph axis is required, particularly at the level of GHRH-R. Two- and 18-month-old male Sprague Dawley rats were treated for 14 days with a daily sc injection of 0.5 or 1.0 mg/kg BW human GHRH-(1–29)NH2 or saline. In 2-month-old rats, treatment with 0.5 mg/kg GHRH increased the number of high affinity pituitary GHRH-R-binding sites by 2-fold (P < 0.05) and hypothalamic somatostatin (SRIF) content by 45% (P < 0.05). It did not affect hypothalamic GHRH content, serum total insulin-like growth factor I (IGF-I), or body weight gain. Treatment with 1.0 mg/kg GHRH decreased the number of high affinity pituitary GHRH-R-binding sites by 2.4-fold compared with that in rats treated with 0.5 mg/kg BW (P < 0.05) and increased hypothalamic SRIF content by 45% (P < 0.05), but did not affect GHRH content. It also decreased circulating levels of IGF-I by 13% (P < 0.05) and slowed the growth rate by 17% (P < 0.05). In 18-month-old rats, treatment with 0.5 mg/kg GHRH for 14 days was not sufficient to rejuvenate pituitary GHRH binding parameters. However, treatment with 1.0 mg/kg GHRH restored the affinities of high and low affinity classes of GHRH-binding sites to values similar to those found in 2-month-old rats. Binding capacities of the high and low affinity classes of sites were increased by 1.8- and 3-fold, respectively, although significance was only reached for the low affinity site (P < 0.05). These changes were associated with a normalization of the level of 2.5-kb GHRH-R messenger RNA transcript, which was decreased by 31% in aging rats (P < 0.05), and by a trend for an increase in the 4-kb GHRH-R messenger RNA transcript, which was already increased by 49% in 18-month-old rats (P < 0.05). A normalization of serum IGF-I levels, which were decreased by 11% in 18-month-old control rats (P < 0.01), was also observed. No treatment effect was detected on body weight or hypothalamic SRIF and GHRH contents. We conclude that a 14-day administration of GHRH induces a differential GHRH-R-mediated regulation at the level of the pituitary and probably the hypothalamus as a function of age.

Can GHRH or GH secretagogues re-initiate pituitary GH pulsatility?

Can GHRH or GH secretagogues re-initiate pituitary GH pulsatility?

Somatostatin decreases somatostatin messenger ribonucleic acid levels in the rat periventricular nucleus

Growth hormone (GH) secretion from the pituitary is known to be under the dual control of GH-releasing factor (GRF) and somatostatin (SRIF). Hypothalamic SRIF, the major inhibitor of pituitary growth hormone secretion, inhibits its own release by a negative ultrashort-loop feedback mechanism. However, it is not known whether this negative regulation is mediated by inhibition of SRIF mRNA production. GRF may also inhibit its own release, thereby modifying pituitary GH secretion, possibly through an ultrashort-loop feedback mechanism. Thus, SRIF production and GRF release are both regulated by SRIF. Periventricular nucleus (PeN) and mediobasal hypothalamus (MBH) from adult male rats were incubated for 6 h in Waymouth’s medium with either SRIF or the SRIF agonist analog RC 160 (10 −9 to 10 −6 M). Levels of SRIF mRNA were determined by an S1 nuclease protection assay using a 32[P]-labeled rat SRIF riboprobe. SRIF (10 −7 M) and RC 160 (10 −8, 10 −7 M) significantly ( p ≤ 0.01) decreased SRIF mRNA levels in the PeN. The levels of SRIF mRNA in the MBH were not modified by either SRIF or RC 160. SRIF (10 −7 and 10 −6 M) significantly ( p ≤ 0.01 and p ≤ 0.001, respectively) inhibited the release of GRF at 30 min in the MBH. Likewise, the release of GRF was slightly decreased by 10 −7 M RC 160, and significantly inhibited by 10 −6 M ( p ≤ 0.001) at 30 min. At 6 h, the levels of GRF were significantly reduced by 10 −7 M SRIF ( p ≤ 0.05) and by RC 160 (10 −7, 10 −6 M; p ≤ 0.001 and p ≤ 0.05, respectively). In contrast with these results, the SRIF analog was unable to alter SRIF release at 30 min. At 6 h incubation, RC 160 (10 −7 M) significantly ( p ≤ 0.001) reduced SRIF release from MBH fragments. These results demonstrate that SRIF and a SRIF analog decrease SRIF mRNA levels in the PeN and inhibit the release of SRIF from the nerve terminals of the MBH. Thus, SRIF appears to regulate its own gene expression by negative ultrashort-loop feedback. Therefore, when SRIF is secreted from these neurons in response to GRF, it down-regulates the preceding stimulatory input as well as its own secretion.

Effects of GH and IGF-I administration on GHRH and somatostatin mRNA levels: I. A study on ad libitum fed and starved adult male rats.

The individual role played by GH and IGF-I in the regulation of hypothalamic GHRH and SRIF gene expression is still object of debate. We have investigated the effect of exogenously administered recombinant hGH (rhGH) and recombinant hIGF-I (rhIGF-I) in ad libitum fed control and starved rats, the latter an animal model which is characterized by low circulating levels of endogenous GH and IGF-I. Adult male rats were fed ad libitum (C) or food-deprived (S) for 72 hours; rats in either C or S groups were treated with systemic administration of rhGH and rhIGF-I for 3 days. GHRH, SRIF and GH mRNA levels were evaluated by Northern and slot blot hybridization. Administration of rhGH (250 micrograms/kg/twice daily, sc) induced a significant inhibition of GHRH and a significant stimulation of SRIF mRNA levels in C rats; GH treatment was, however, ineffective on both neuropeptide mRNA levels in the S group. Continuous infusion of rhIGF-I (300 micrograms/kg/day, sc) induced a significant increase of SRIF levels in both C and S rats but did not modify GHRH mRNA levels in either group. In the pituitary, GH mRNA levels followed a pattern very similar to that of GHRH. These results provide evidence for a direct role of GH in the inhibition of GHRH mRNA levels; IGF-I appears more involved in the direct stimulation of SRIF mRNA levels.

Negative regulation of hypothalamic growth hormone-releasing factor messenger ribonucleic acid by growth hormone and insulin-like growth factor I.

Increased growth hormone-releasing factor messenger ribonucleic acid (GRF mRNA) and decreased somatostatin (SRIF) mRNA levels have been reported in the hypothalamus of hypophysectomized rats as well as of dwarf mice. In order to elucidate the effect of the growth hormone-insulin-like growth factor I (GH-IGF-I) axis on hypothalamic GRF and SRIF synthesis, we measured levels of mRNA coding for GRF and SRIF and for pituitary GH in pubertal male rats treated for 3 weeks with antirat GRF gamma-globulin (GRF-ab), anti-SRIF gamma-globulin (SRIF-ab) or both. Immunoneutralization of circulating endogenous GRF resulted in a marked decrease in serum IGF-I and pituitary GH mRNA levels in Northern blot analysis, whereas it caused a significant increase in GRF mRNA levels in the arcuate nucleus as assessed by both Northern blot and in situ hybridization analysis. SRIF mRNA levels in the periventricular nucleus were slightly decreased by GRF-ab treatment when analyzed by in situ hybridization, but not significantly after Northern blot analysis. Immunoneutralization of circulating endogenous SRIF failed to affect mRNA levels of hypothalamic GRF and SRIF but caused a slight reduction in pituitary GH mRNA levels. Levels of mRNA coding for hypothalamic GRF and pituitary GH were also measured by Northern blot analysis in young male rats treated with rat GRF-ab for 2 weeks and replaced with rat GH or IGF-I for the second 1 week. Replacement with either rat GH or IGF-I suppressed the increased hypothalamic GRF mRNA levels. These data indicate that endogenous GRF is essential for normal synthesis of pituitary GH and that both GH and IGF-I negatively regulate the synthesis of hypothalamic GRF.

Regulation of somatostatin synthesis by GABA A receptor stimulation in mouse brain

Neuroanatomical data have documented the existence of synaptic contacts between γ-aminobutyric acid (GABA) terminals and periventricular hypothalamic somatostatin (SRIF) neurons. In other brain regions, like the cortex or hippocampus, GABA and SRIF are colocalized in short interneurons. These observations suggest that GABA modulates SRIF neuronal activity. In order to test this hypothesis, we studied the effects of the in vivo stimulation of the GABA A receptor (muscimol, 0.75 mg/kg + diazepam, 2.5 mg/kg) on SRIF content and preproSRIF mRNA levels, in mouse brain. Chronic (7 days), but not acute, treatment induced a 38% decrease in hypothalamic SRIF content (as estimated by RIA), a 20% decrease in cortex and no effect in the striatum. The decrease in hypothalamic and cortical SRIF levels lasted until 24 h after cessation of the treatment. In the hypothalamus, prosomatostatin mRNA levels were estimated by Northern blot analysis using a 32P-labeled 45-mere oligoprobe. ProSRIF mRNA hypothalamic levels were equally (48%) decreased by the acute and chronic treatments and remained lower than controls 48 h after the last injection. Quantitative in situ hybridization was used to examine the regional distribution of GABA-induced acute inhibition of proSRIF mRNA densities, using the same oligomere labeled with 35S. ProSRIF mRNA levels were decreased by 35% in the periventricular hypothalamic nucleus. In contrast, no significant modification was observed in cortex, striatum and hilus of the dentate gyrus of the dorsal hippocampus. The present data demonstrate a regionally selective inhibitory action of GABA, mediated by GABA A receptors stimulation, on the biosynthetic mechanisms of the long projecting neuroendocrine SRIF neurons of the anterior periventricular nucleus of the hypothalamus.

Hypothalamic preprosomatostatin messenger ribonucleic acid expression in mice transgenic for excess or deficient endogenous growth hormone.

The influence of altered endogenous GH status on somatostatin (somatotropin release-inhibiting hormone; SRIF) gene expression was studied in two transgenic mouse models. Transgenic dwarf mice carried the rat GH gene promoter fused to the diphtheria toxin A-chain gene, placing toxin expression under GH promoter control. As a result, the toxic product of the transgene ablated all GH-expressing cells, resulting in undetectable circulating GH, reduced weight (10.6 +/- 1.0 g for transgenic dwarfs vs. 29.5 +/- 1.7 g for controls; P less than 0.001), and no detectable somatotrophs. Transgenic giant mice contained a construction combining a widely expressed metallothionein promoter and the human GH-releasing hormone (hGHRF) structural gene. Transgene expression of hGHRF resulted in overproduction of endogenous mouse GH in the anterior pituitary and weight increases (42.7 +/- 2.7 g for giants vs. 29.5 +/- 1.7 g for controls; P less than 0.005). Using in situ hybridization, control mice, transgenic dwarfs, and transgenic giants were compared for levels of prepro-SRIF mRNA. Hybridization signal intensities for prepro-SRIF mRNA were similar in transgenic dwarfs to those in littermate nontransgenic mice in non-GH-regulating regions of the brain, such as cortex (control, 31 +/- 2 U; dwarf, 27 +/- 2) and reticulothalamic nucleus (control, 41 +/- 2 U; dwarfs, 39 +/- 3). Transgenic giant mice had hybridization intensity of SRIF mRNA similar to that of normals in cortex (controls, 31 +/- 2 U; giant, 27 +/- 1) and reticulothalamic nucleus (controls, 41 +/- 2 U; giant, 40 +/- 4). In the GH-regulating neurons of the anterior periventricular hypothalamus (PeN), prepro-SRIF mRNA signal in transgenic dwarf mice decreased to 60% of that in controls (88 +/- 13 U for dwarfs vs. 147 +/- 17 U for controls; P less than 0.01), although the numbers of mRNA-expressing cells in the PeN were not different between the transgenic dwarfs and controls (dwarfs, 69 +/- 6 cells; controls, 72 +/- 4 cells). The transgenic giant mice had 230% higher prepro-SRIF mRNA signal than control mice in the PeN (343 +/- 30 U in giants vs. 147 +/- 17 U in controls; P less than 0.001). Again, the numbers of mRNA-expressing cells were not different in giants (57 +/- 9) and normals (72 +/- 4). These results suggest that while the lack of endogenous GH is accompanied by a slight decrease in transcriptional expression of SRIF in the PeN, the overproduction of endogenous GH greatly stimulates hypothalamic SRIF steady state mRNA levels.

Regulation of Growth Hormone-releasing Hormone Gene Expression and Secretion

Growth hormone (GH)-releasing hormone (GRH) is a stimulatory hypothalamic hypophysiotropic hormone which, along with an inhibitory peptide, somatostatin (SRIF), regulates the synthesis and secretion of GH in anterior pituitary somatotrophs. Although GHRH genes in several species have been characterized, there is only a limited understanding of the neural and hormonal mechanisms regulating GRH biosynthesis and secretion. Recent progress in PCR and in situ hybridization techniques as well as hGRF-transgenic animal models have provided an opportunity to study the regulation of GRH gene expression and secretion as well as its metabolism. The difference in 5'-untranslated sequences in both mouse and rat GRH cDNAs from hypothalamus and placenta has also suggested tissue-specific regulation of the GRH gene. GH excess has been shown to result in a decrease in hypothalamic GRH mRNA as well as GRH content and secretion while GH deficiency caused by hypophysectomy, hypothyroidism or genetic dwarfism causes an increase in GRH mRNA levels as tested by Northern blot analysis or in situ hybridization. Treatment of animals with GH or SRIF inhibits the increased GRH gene expression in the hypothalamic arcuate nucleus. Double immunocytochemistry for hypothalamic GRH and SRIF has shown both axo-perikaryal and axo-axonal connections between GRH- and SRIF- containing neurons. SRIF binding and GH receptor mRNA are demonstrated on a subpopulation of GRH-containing neurons in the hypothalamic arcuate nucleus. It is therefore possible to conclude that regulation of GRH gene expression, primarily related to inhibitory feedback effects of GH and IGFs on hypothalamic GRH gene expression, is mediated at least in part by SRIF or GH. The single transcript of the human GRH gene encodes a 108 amino acid precursor, prepro-hGRH, which is cleaved into the signal peptide and the remaining peptide, pro-hGRH. The latter is further processed to yield two equipotent forms of the releasing hormone, hGRH(1-44)-NH2, hGRH(1-40)-OH, and a carboxyl-terminal peptide (hGCTP) of unknown function. Studies in transgenic mice demonstrate the processing of hGRH-prohormone into both mature forms of hGRH and hGCTP, and provide evidence that hGRH(1-40)-OH is derived from hGRH(1-44)-NH2.(ABSTRACT TRUNCATED AT 400 WORDS)

A hypothalamic hormone-somatostatin--from endocrinology to neurophysiology

The densest distribution of somatostatin (SRIF) neuron perikarya is localized in the hypothalamic periventricular nucleus (Pe) close to the third ventricle, from which many fibers are projected to the median eminence. The release of SRIF in the neurohemal organ into the anterior pituitary modulates GH secretion from pituitary somatotrophs. When SRIF input from the hypothalamus to rat anterior pituitary is reduced by either neurosurgery or SRIF antiserum iv injection, the responsiveness of the pituitaries to human GH releasing factor (hGRF) in an in vitro perifusion system is markedly attenuated. Moreover, SRIF pretreatment facilitates the GH release response of dispersed anterior pituitary cells to hGRF. The long lasting SRIF effect to sensitize somatotrophs appears to take place beyond cAMP formation or as an unknown distal effect. These findings indicate that SRIF neurons in the Pe play a role in maintaining the pituitary responsiveness to GRF in addition to the original action to inhibit GH secretion. Neuronal networks between Pe-SRIF neurons, and intra- and extrahypothalamic nuclei are identified by Pe stimulation test on GRF-GH secretion. In addition to the physiological role in maintaining pituitary responsiveness, Pe SRIF neurons have a wide influence on specific SRIF receptor binding in various brain regions as well as in the anterior pituitary. Shortly after lesioning the Pe neurons, there is a continuous increase in plasma GH level with a transient increase in specific binding of 125I-Tyr 11-SRIF-14 to the anterior pituitary. Furthermore, there is a similar but a little longer increase in binding of the radioligand to some brain areas such as the cerebral cortex, hippocampus, and amygdala nuclei. However, neuronal connections between the SRIF neurons and nuclei which are up-regulated by the lesioning have not been fully proven. When the labeled ligand is infused into the lateral ventricle, it is rapidly and widely distributed in many periventricular structures in the lateral and third ventricles. These findings suggest that SRIF produced in the Pe neurons is transported to other brain areas via cerebrospinal fluid in addition to neuronal connections for modulating the activity of neurons which have SRIF receptors. Thus, hypothalamic Pe SRIF neurons have dualistic roles for controlling anterior pituitary function and modulating CNS neuron activity.

Short-term adult exposure to estradiol feminizes the male pattern of spontaneous and growth hormone-releasing factor-stimulated growth hormone secretion in the rat

Experimental evidence indicates that the neonatal gonadal steroid environment is an important determinant of the sexual dimorphism of GH secretion and body growth. However, the influence of the sex steroids in GH control during adult life and their mechanism/site of action are largely unknown. In the present study we examined the effects of adult gonadectomy (GNX) and short term adult exposure to 17 beta-estradiol (E2) on both spontaneous and GRF-stimulated GH release in free-moving adult male rats. The rate of body weight gain was also monitored. GNX (3 weeks postoperatively) resulted in a 2-fold reduction in GH pulse amplitude compared to that in sham-operated control rats, but did not significantly alter the GH nadir or the interpeak interval. Exposure to E2 (sc implants) for 4 days markedly disrupted the spontaneous GH secretory profile of both sham-operated and GNX rats; E2-treated animals exhibited a striking elevation (4- to 20-fold) of GH trough levels and a significant decrease in GH interpeak interval, remarkably similar to the typical female rat GH secretory profile. The augmentation in both GH nadir and GH pulse frequency was evident as early as 12 h after a single sc injection of E2 valerate. In sham and GNX rats bearing control implants, the GH response to 1 micrograms rat GRF-(1-29)NH2, iv, was significantly greater when GRF was administered at peak (1100 h) than at trough (1300 h) times of GH secretion; the latter is known to be due to antagonism by the cyclical increased release of endogenous somatostatin (SRIF) in the male rat. Treatment with E2 abolished this time-dependent difference in both groups and produced a regular pattern of GH responsiveness to GRF similar to that typically observed in the female rat, thus suggesting that E2 has altered the pattern of hypothalamic SRIF secretion from a cyclic to a more continuous mode of release. Chronic exposure to E2 for 2 weeks resulted in an almost 6-fold inhibition of the rate of body weight gain in sham-operated male rats to levels comparable to those in normal adult female rats. Taken together, these results demonstrate that short term exposure to E2 during adult life can profoundly feminize the male pattern of spontaneous and GRF-stimulated GH secretion, as well as rate of somatic growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypothalamic regulation of impaired growth hormone secretion in diabetic rats. 2. Studies in spontaneously diabetic BB Worcester rats.

In the present study we investigated the effects of modulating endogenous somatostatin (SRIF) on the GH response to growth hormone-releasing hormone (GHRH) in spontaneously diabetic BB/Wor rats and nondiabetic littermates. Plasma growth hormone (GH) concentrations following injection of GHRH (500 ng/kg, i.v.) were measured in the rats after pretreatment with either normal sheep serum+saline (NSS+SAL), somatostatin antibody (SRIF-Ab), or pyridostigmine bromide (PD), an acetylcholine esterase inhibitor hypothesized to decrease hypothalamic SRIF tone. The GH response to GHRH in spontaneous diabetic rats pretreated with NSS+SAL was significantly lower (p less than 0.05) than the response observed in the nondiabetic group. SRIF-Ab pretreatment reversed the blunted GH response observed in the diabetic rats. However, PD pretreatment was not effective. These results indicate that the blunted GH response observed in BB/Wor diabetic rats is reversed by neutralization of endogenous SRIF with SRIF-Ab and leads to the conclusion that SRIF plays an active role in modulating GH secretion in spontaneously diabetic rats. The failure of PD to modulate the GH response suggests this acetylcholine agonist is ineffective in this animal paradigm.

Hypothalamic regulation of impaired growth hormone secretion in diabetic rats. 1. Studies in streptozotocin-induced diabetic rats.

Growth hormone (GH) secretion is blunted in diabetic rats. In the present experiment we observed that pituitary GH concentrations and the plasma GH response to an exogenous dose of growth hormone-releasing hormone (GHRH) is decreased in streptozotocin-induced diabetic rats (p less than 0.02) with respect to normal rats. In an attempt to determine if increased somatostatin (SRIF) secretion is responsible for the decreased GH secretion, we studied the effect of modulating SRIF tone on the GH response to GHRH in normal and streptozotocin-induced diabetic rats. Rats were pretreated with either normal sheep serum and saline (NSS+SAL), somatostatin antibodies (SRIF-Ab), or pyridostigmine (PD), an acetylcholinesterase inhibitor hypothesized to reduce hypothalamic SRIF secretion. Pretreatment of normal rats with SRIF-Ab or PD resulted in an increased GH response to exogenous GHRH in comparison to NSS+SAL-pretreated normal rats at 5 min postinjection. In contrast, pretreatment of diabetic rats with SRIF-Ab or PD did not alter the GH response to exogenous GHRH when compared to NSS+SAL-pretreated diabetic animals. These results suggest that the blunted GH response to exogenous GHRH observed in streptozotocin-induced diabetic rats may not be due to an increase of endogenous SRIF tone.

Short-term adult exposure to estradiol feminizes the male pattern of spontaneous and growth hormone-releasing factor-stimulated growth hormone secretion in the rat.

Experimental evidence indicates that the neonatal gonadal steroid environment is an important determinant of the sexual dimorphism of GH secretion and body growth. However, the influence of the sex steroids in GH control during adult life and their mechanism/site of action are largely unknown. In the present study we examined the effects of adult gonadectomy (GNX) and short term adult exposure to 17 beta-estradiol (E2) on both spontaneous and GRF-stimulated GH release in free-moving adult male rats. The rate of body weight gain was also monitored. GNX (3 weeks postoperatively) resulted in a 2-fold reduction in GH pulse amplitude compared to that in sham-operated control rats, but did not significantly alter the GH nadir or the interpeak interval. Exposure to E2 (sc implants) for 4 days markedly disrupted the spontaneous GH secretory profile of both sham-operated and GNX rats; E2-treated animals exhibited a striking elevation (4- to 20-fold) of GH trough levels and a significant decrease in GH interpeak interval, remarkably similar to the typical female rat GH secretory profile. The augmentation in both GH nadir and GH pulse frequency was evident as early as 12 h after a single sc injection of E2 valerate. In sham and GNX rats bearing control implants, the GH response to 1 micrograms rat GRF-(1-29)NH2, iv, was significantly greater when GRF was administered at peak (1100 h) than at trough (1300 h) times of GH secretion; the latter is known to be due to antagonism by the cyclical increased release of endogenous somatostatin (SRIF) in the male rat. Treatment with E2 abolished this time-dependent difference in both groups and produced a regular pattern of GH responsiveness to GRF similar to that typically observed in the female rat, thus suggesting that E2 has altered the pattern of hypothalamic SRIF secretion from a cyclic to a more continuous mode of release. Chronic exposure to E2 for 2 weeks resulted in an almost 6-fold inhibition of the rate of body weight gain in sham-operated male rats to levels comparable to those in normal adult female rats. Taken together, these results demonstrate that short term exposure to E2 during adult life can profoundly feminize the male pattern of spontaneous and GRF-stimulated GH secretion, as well as rate of somatic growth.(ABSTRACT TRUNCATED AT 400 WORDS)