Stimulation of GHRH Neuron Axon Growth by Leptin and Impact of Nutrition during Suckling in Mice.

Nutrition during the early postnatal period can program the growth trajectory and adult size. Nutritionally regulated hormones are strongly suspected to be involved in this physiological regulation. Linear growth during the postnatal period is regulated by the neuroendocrine somatotropic axis, whose development is first controlled by GHRH neurons of the hypothalamus. Leptin that is secreted by adipocytes in proportion to fat mass is one of the most widely studied nutritional factors, with a programming effect in the hypothalamus. However, it remains unclear whether leptin stimulates the development of GHRH neurons directly. Using a Ghrh-eGFP mouse model, we show here that leptin can directly stimulate the axonal growth of GHRH neurons in vitro in arcuate explant cultures. Moreover, GHRH neurons in arcuate explants harvested from underfed pups were insensitive to the induction of axonal growth by leptin, whereas AgRP neurons in these explants were responsive to leptin treatment. This insensitivity was associated with altered activating capacities of the three JAK2, AKT and ERK signaling pathways. These results suggest that leptin may be a direct effector of linear growth programming by nutrition, and that the GHRH neuronal subpopulation may display a specific response to leptin in cases of underfeeding.

GHRH expression plasmid improves osteoporosis and skin damage in aged mice.

The hormone secretion of GHRH-GH-IGF-1 axis in animals was decreased as aging. These hormones play an important role in maintaining bone mass and bone structure, and also affect the normal structure and function of the skin. We used plasmid-based technology to deliver growth hormone releasing hormone (GHRH) to elderly mice. In the current study, 80 and 120 µg/kg pVAX-GHRH plasmid expression plasmid were injected into old mice, the serum GHRH and insulin-like growth factor-1(IGF-1) content were increased within three weeks (P < 0.05). In the groups of 80 and 120 µg/kg plasmid, the content of procollagen type I N-terminal pro-peptide (PINP) in the serum was increased(P < 0.05), and the content of C-terminal telopeptides of type I collagen (CTX-1) in the serum was reduced significantly (P < 0.05). Furthermore, the expression of osteoprotegerin (OPG) and osteocalcin (OCN) in the femur also was increased(P < 0.05). The bone mineral density(BMD)、trabecular bone volume (BV/TV) and trabecular number(Tb.N) of mouse femur were increased significantly (P < 0.05) and trabecular separation(Tb.Sp) was decreased(P < 0.05). There were more trabecular bones in the bone marrow cavity and the trabecular bones are thicker in the groups of 80 and 120 µg/kg plasmid relative to control. The superoxide dismutase (SOD) content in the skin was increased(P < 0.05), and the malondialdehyde (MDA) content was reduced significantly (P < 0.05). Meanwhile, the skin moisture content also increased significantly(P < 0.05). Moreover, the expression of matrix metalloproteinase 3(MMP3) and matrix metalloproteinase 9(MMP9) was decreased in the skin(P < 0.05). The thickness of the dermis and epidermis of the skin had increased significantly(P < 0.05). Skin structure is more dense and complete in the two groups. These results indicate that 80 and 120 µg/kg plasmid-mediated GHRH supplementation can improve osteoporosis and skin aging in aged mice.

Growth hormone-releasing hormone antagonistic analog MIA-690 stimulates food intake in mice.

In addition to its metabolic and endocrine effects, growth hormone-releasing hormone (GHRH) was found to modulate feeding behavior in mammals. However, the role of recently synthetized GHRH antagonist MIA-690 and MR-409, a GHRH agonist, on feeding regulation remains to be evaluated. We investigated the effects of chronic subcutaneous administration of MIA-690 and MR-409 on feeding behavior and energy metabolism, in mice. Compared to vehicle, MIA-690 increased food intake and body weight, while MR-409 had no effect. Both analogs did not modify locomotor activity, as well as subcutaneous, visceral and brown adipose tissue (BAT) mass. A significant increase of hypothalamic agouti-related peptide (AgRP) gene expression and norepinephrine (NE) levels, along with a reduction of serotonin (5-HT) levels were found after MIA-690 treatment. MIA-690 was also found able to decrease gene expression of leptin in visceral adipose tissue. By contrast, MR-409 had no effect on the investigated markers. Concluding, chronic peripheral administration of MIA-690 could play an orexigenic role, paralleled by an increase in body weight. The stimulation of feeding could be mediated, albeit partially, by elevation of AgRP gene expression and NE levels and decreased 5-HT levels in the hypothalamus, along with reduced leptin gene expression, in the visceral adipose tissue.

Substance P appears to affect growth via growth hormone-releasing hormone (GHRH) neurons in the human hypothalamus.

Substance P is an eleven-amino acid neuropeptide (undecapeptide) with multiple effects on the gastrointestinal, cardiovascular, and urinary systems as well as complex central nervous system functions such as pain, learning, memory, and sexual homeostasis. Previous studies also revealed that substance P exhibits regulatory effects on growth possibly via influencing hypothalamic GHRH release in human. However, the morphological substrate of this phenomenon has not been elucidated yet. In the present study, we examined the putative presence of juxtapositions between the substance P- and GHRH-immunoreactive (IR) systems using double-label immunocytochemistry. High-magnification light microscopy with oil immersion was used to identify putative juxtapositions between these systems. Our studies revealed substance P-IR fiber network abutting on the surface of the majority of GHRH-immunoreactive neurons in the human hypothalamus. These fiber varicosities often cover a significant surface area on the GHRH-IR neurons, forming basket-like encasements with multiple en passant type contacts. The majority of these densely innervated GHRH-IR neurons were found in the infundibular nucleus/median eminence, while substance P-IR fibers often abut on the GHRH-IR neurons in the periventricular zone and basal perifornical area of the tuberal region and in the dorsomedial subdivision of the ventromedial nucleus. The posterior hypothalamus did not contain observable substance P-GHRH associations. The density and the morphology of these intimate associations suggest that substance P influences growth by regulating hypothalamic GHRH release by direct synaptic contacts.

Specific subpopulations of hypothalamic leptin receptor-expressing neurons mediate the effects of early developmental leptin receptor deletion on energy balance.

OBJECTIVE: To date, early developmental ablation of leptin receptor (LepRb) expression from circumscribed populations of hypothalamic neurons (e.g., arcuate nucleus (ARC) Pomc- or Agrp-expressing cells) has only minimally affected energy balance. In contrast, removal of LepRb from at least two large populations (expressing vGat or Nos1) spanning multiple hypothalamic regions produced profound obesity and metabolic dysfunction. Thus, we tested the notion that the total number of leptin-responsive hypothalamic neurons (rather than specific subsets of cells with a particular molecular or anatomical signature) subjected to early LepRb deletion might determine energy balance. METHODS: We generated new mouse lines deleted for LepRb in ARC GhrhCre neurons or in Htr2cCre neurons (representing roughly half of all hypothalamic LepRb neurons, distributed across many nuclei). We compared the phenotypes of these mice to previously-reported models lacking LepRb in Pomc, Agrp, vGat or Nos1 cells. RESULTS: The early developmental deletion of LepRb from vGat or Nos1 neurons produced dramatic obesity, but deletion of LepRb from Pomc, Agrp, Ghrh, or Htr2c neurons minimally altered energy balance. CONCLUSIONS: Although early developmental deletion of LepRb from known populations of ARC neurons fails to substantially alter body weight, the minimal phenotype of mice lacking LepRb in Htr2c cells suggests that the phenotype that results from early developmental LepRb deficiency depends not simply upon the total number of leptin-responsive hypothalamic LepRb cells. Rather, specific populations of LepRb neurons must play particularly important roles in body energy homeostasis; these as yet unidentified LepRb cells likely reside in the DMH.

Hypothalamic abnormalities: Growth failure due to defects of the GHRH receptor.

Several acquired or congenital hypothalamic abnormalities may cause growth failure (GF). We described two of these congenital abnormalities. First, a case of CHARGE syndrome, an epigenetic disorder mostly caused by heterozygous mutations in the gene encoding CHD7, a chromatin remodeling protein, causing several malformations, some life-threatening, with additional secondary hypothalamus-hypophyseal dysfunction, including GF. Second, a cohort of individuals with genetic isolated severe GH deficiency (IGHD), due to a homozygous mutation in the GH-releasing hormone (GHRH) receptor gene described in Itabaianinha County, in northeast Brazil. In this IGHD, with marked reduction of serum concentrations of IGF-I, and an up regulation of IGF-II, GF is the principal finding in otherwise normal subjects, with normal quality of life and longevity. This IGHD may unveil the effects of GHRH, pituitary GH and IGF-I, IGF-II and local GH and growth factor on the size and function of body and several systems. For instance, anterior pituitary hypoplasia, and impairment of the non-REM sleep may be due to GHRH resistance. Proportionate short stature, doll facies, high-pitched pre-pubertal voice, and reduced muscle mass reflect the lack of the synergistic effect of pituitary GH and IGF-I in bones and muscles. Central adiposity may be due to a direct effect of the lack of GH. Brain, eyes and immune system may also involve IGF-II and local GH or growth factors. A concept of physiological hierarchy controlling body size and function by each component of the GH system may be drawn from this model.

LXR activation increases the expression of GnRH AND αMSH in the rat hypothalamus in vivo.

Liver X receptors (LXR) are important transcription factors involved in the regulation of carbohydrate and lipid metabolism. Recently, we described LXR receptors expression in the hypothalamus but their function in this brain area remains unknown. Here, we evaluated the function of LXR on the expression of factors produced in the hypothalamus in vitro and in vivo by Western blotting and immunocytochemistry. More precisely we studied the expression of GnRH and GHRH, αMSH and NPY in male Sprague-Dawley rats. The effects of two synthetic LXR agonists, T0901317 and GW3965, were first tested in vitro. Hypothalamic explants were treated with either T0901317 or GW3965 (10µM) for 2, 4, 6 and 8h. As a positive control the cholesterol ABCA1 and glucose GLUT2 transporters were used. No changes were observed in the expression of the factors evaluated in vitro. The effects of the LXR agonists were then tested in vivo. Rats were injected ICV into the third ventricle with either T0901317 or GW3965 (2.5µg/5µL ICV) and after 3.5h or 24h the hypothalami were dissected out and rapidly frozen for analysis. αMSH and GnRH expression was significantly increased after 3.5h of T0901317 treatment. Anterior/posterior hypothalamic ratio increases for αMSH expression and decreases for GnRH expression after 24h of LXR activation. Altogether these results show that LXR activation affects the expression of GnRH and αMSH, suggesting that LXR in the hypothalamus is capable of modulating hypothalamic responses related to appetite, sexual behavior and reproductive functions.

Corticotropin-releasing hormone (CRH)-immunoreactive (IR) axon varicosities target a subset of growth hormone-releasing hormone (GHRH)-IR neurons in the human hypothalamus.

It is a general consensus that stress is one of the major factors that suppresses growth. Previous studies revealed that the catecholaminergic and neuropeptide Y (NPY) systems, involved in the activation of stress-related neuronal circuits, influence growth hormone (GH)-release via modulating growth hormone-releasing hormone (GHRH) secretion. Indeed, catecholaminergic and NPY-immunoreactive (IR) axon varicosities abut on the surface of the GHRH neurons forming contacts. These juxtapositions appear to be real synapses and may represent the morphological substrate of the impact of stress on growth. In addition to catecholamines and NPY, there is a vast amount of evidence that corticotropin-releasing hormone (CRH), a major stress hormone, also influences GH secretion. Whether this modulatory effect is direct, or indirect, via the hypothalamic GHRH system, has not been elucidated yet. In the present study, we examined the possibility that CRH influences GH secretion via modulating the GHRH release by direct synaptic mechanisms. Since the verification of these synapses by electron microscopy is problematic in human due to the long post mortem time, in order to reveal the putative CRH-GHRH juxtapositions, light microscopic double label immunohistochemistry was utilized. In the infundibular nucleus, a subset (6%) of the GHRH perikarya received abutting CRH fiber varicosities forming multiple contacts while passing by. No gaps appeared between the contacting elements. The morphology of these CRH-GHRH juxtapositions suggests that, among other neurotransmitters/neuromodulators, CRH influences growth by modulating the hypothalamic GHRH secretion via direct synaptic mechanisms.

Hypothalamic neurogenesis persists in the aging brain and is controlled by energy-sensing IGF-I pathway.

Hypothalamic tanycytes are specialized glial cells lining the third ventricle. They are recently identified as adult stem and/or progenitor cells, able to self-renew and give rise to new neurons postnatally. However, the long-term neurogenic potential of tanycytes and the pathways regulating lifelong cell replacement in the adult hypothalamus are largely unexplored. Using inducible nestin-CreER(T2) for conditional mutagenesis, we performed lineage tracing of adult hypothalamic stem and/or progenitor cells (HySC) and demonstrated that new neurons continue to be born throughout adult life. This neurogenesis was targeted to numerous hypothalamic nuclei and produced different types of neurons in the dorsal periventricular regions. Some adult-born neurons integrated the median eminence and arcuate nucleus during aging and produced growth hormone releasing hormone. We showed that adult hypothalamic neurogenesis was tightly controlled by insulin-like growth factors (IGF). Knockout of IGF-1 receptor from hypothalamic stem and/or progenitor cells increased neuronal production and enhanced α-tanycyte self-renewal, preserving this stem cell-like population from age-related attrition. Our data indicate that adult hypothalamus retains the capacity of cell renewal, and thus, a substantial degree of structural plasticity throughout lifespan.

Melatonin modulates monochromatic light-induced GHRH expression in the hypothalamus and GH secretion in chicks.

To study the mechanism by which monochromatic lights affect the growth of broilers, a total of 192 newly hatched broilers, including the intact, sham-operated and pinealectomy groups, were exposed to white light (WL), red light (RL), green light (GL) and blue light (BL) using a light-emitting diode (LED) system for 2 weeks. The results showed that the GHRH-ir neurons were distributed in the infundibular nucleus (IN) of the chick hypothalamus. The mRNA and protein levels of GHRH in the hypothalamus and the plasma GH concentrations in the chicks exposed to GL were increased by 6.83-31.36%, 8.71-34.52% and 6.76-9.19% compared to those in the chicks exposed to WL (P=0.022-0.001), RL (P=0.002-0.000) and BL (P=0.290-0.017) in the intact group, respectively. The plasma melatonin concentrations showed a positive correlation with the expression of GHRH (r=0.960) and the plasma GH concentrations (r=0.993) after the various monochromatic light treatments. After pinealectomy, however, these parameters decreased and there were no significant differences between GL and the other monochromatic light treatments. These findings suggest that melatonin plays a critical role in GL illumination-enhanced GHRH expression in the hypothalamus and plasma GH concentrations in young broilers.

Changes of growth hormone-releasing hormone and somatostatin neurons in the rat hypothalamus induced by genistein: a stereological study.

OBJECTIVES: Genistein is a plant-derived estrogenic isoflavone commonly found in dietary and therapeutic supplements, due to its potential health benefits. Growth hormone-releasing hormone (GHRH) and somatostatin (SS) are neurosecretory peptides synthesized in neurons of the hypothalamus and regulate the growth hormone secretion. Early reports indicate that estrogens have highly involved in the regulation of GHRH and SS secretions. Since little is known about the potential effects of genistein on GHRH and SS neurons, we exposed rats to genistein. METHODS: Genistein were administered to adult rats in dose of 30 mg/kg, for 3 weeks. The estradiol-dipropionate treatment was used as the adequate controls to genistein. Using applied stereology on histological sections of hypothalamus, we obtained the quantitative information on arcuate (Arc) and periventricular (Pe) nucleus volume and volume density of GHRH neurons and SS neurons. Image analyses were used to obtain GHRH and SS contents in the median eminence (ME). RESULTS: Administration of estradiol-dipropionate caused the increase of Arc and Pe nucleus volume, SS neuron volume density, GHRH and SS staining intensity in the ME, when compared with control. Genistein treatment increased: Arc nucleus volume and the volume density of GHRH neurons (by 26%) and SS neurons (1.5 fold), accompanied by higher GHRH and SS staining intensity in the ME, when compared to the orhidectomized group. DISCUSSION: These results suggest that genistein has a significant effect on hypothalamic region, involved in the regulation of somatotropic system function, and could contribute to the understanding of genistein as substance that alter the hormonal balance.

Juxtapositions between the somatostatinergic and growth hormone-releasing hormone (GHRH) neurons in the human hypothalamus.

Somatostatin is a 14-28 amino acid peptide that is located not only in the gastrointestinal system but also in multiple sites of the human brain. The inhibitory effect of somatostatin on the growth hormone (GH) secretion of the pituitary gland is a well-established phenomenon. There is a general consensus that somatostatin is released into the hypophysial portal blood and modulates GH secretion by hormonal action. In the present study, we explored the possibility that in addition to the hormonal route, somatostatin may also influence GH secretion via influencing the growth hormone-releasing hormone (GHRH) secretion by direct contacts that may be functional synapses. Since the verification of these putative synapses by electron microscopy is virtually impossible in humans due to the long post mortem time, in order to reveal the putative somatostatinergic-GHRH juxtapositions, light microscopic double-label immunohistochemistry was utilized. By examining the slides with high magnification, we observed that the vast majority of the GHRH perikarya received contacting somatostatinergic axonal varicosities in the arcuate nucleus. In contrast, GHRH axonal varicosities rarely contacted somatostatinergic perikarya. The morphology and the abundance of somatostatin to GHRH juxtapositions indicate that these associations are functional synapses, and they represent, at least partially, the morphological substrate of the somatostatin-influenced GHRH secretion. Thus, in addition to influencing the GH secretion directly via the hypophysial portal system, somatostatin may also modulate GH release from the anterior pituitary by regulating the hypothalamic GHRH secretion via direct contacts. The rare GHRH to somatostatin juxtapositions indicate that the negative feedback effect of GH targets the somatostatinergic system directly and not via the GHRH system.

Effects of hypothalamic dopamine on growth hormone-releasing hormone-induced growth hormone secretion and thyrotropin-releasing hormone-induced prolactin secretion in goats.

The aim of the present study was to clarify the effects of hypothalamic dopamine (DA) on the secretion of growth hormone (GH) in goats. The GH-releasing response to an intravenous (i.v.) injection of GH-releasing hormone (GHRH, 0.25 µg/kg body weight (BW)) was examined after treatments to augment central DA using carbidopa (carbi, 1 mg/kg BW) and L-dopa (1 mg/kg BW) in male and female goats under a 16-h photoperiod (16 h light, 8 h dark) condition. GHRH significantly and rapidly stimulated the release of GH after its i.v. administration to goats (P < 0.05). The carbi and L-dopa treatments completely suppressed GH-releasing responses to GHRH in both male and female goats (P < 0.05). The prolactin (PRL)-releasing response to an i.v. injection of thyrotropin-releasing hormone (TRH, 1 µg/kg BW) was additionally examined in male goats in this study to confirm modifications to central DA concentrations. The treatments with carbi and L-dopa significantly reduced TRH-induced PRL release in goats (P < 0.05). These results demonstrated that hypothalamic DA was involved in the regulatory mechanisms of GH, as well as PRL secretion in goats.

Intimate associations between the endogenous opiate systems and the growth hormone-releasing hormone system in the human hypothalamus.

Although it is a general consensus that opioids modulate growth, the mechanism of this phenomenon is largely unknown. Since endogenous opiates use the same receptor family as morphine, these peptides may be one of the key regulators of growth in humans by impacting growth hormone (GH) secretion, either directly, or indirectly, via growth hormone-releasing hormone (GHRH) release. However, the exact mechanism of this regulation has not been elucidated yet. In the present study we identified close juxtapositions between the enkephalinergic/endorphinergic/dynorphinergic axonal varicosities and GHRH-immunoreactive (IR) perikarya in the human hypothalamus. Due to the long post mortem period electron microscopy could not be utilized to detect the presence of synapses between the enkephalinergic/endorphinergic/dynorphinergic and GHRH neurons. Therefore, we used light microscopic double-label immunocytochemistry to identify putative juxtapositions between these systems. Our findings revealed that the majority of the GHRH-IR perikarya formed intimate associations with enkephalinergic axonal varicosities in the infundibular nucleus/median eminence, while endorphinergic-GHRH juxtapositions were much less frequent. In contrast, no significant dynorphinergic-GHRH associations were detected. The density of the abutting enkephalinergic fibers on the surface of the GHRH perikarya suggests that these juxtapositions may be functional synapses and may represent the morphological substrate of the impact of enkephalin on growth. The small number of GHRH neurons innervated by the endorphin and dynorphin systems indicates significant differences between the regulatory roles of endogenous opiates on growth in humans.

Neuroendocrine Regulation of Growth Hormone Secretion / 대한소아내분비학회지

The regulation of growth hormone (GH) secretion is, to a larger extent, controlled by three hypothalamic hormones: GH-releasing hormone (GHRH), somatostatin, and ghrelin. Each binds to G protein-linked membrane receptors through which signaling occurs. We used a series of genetic and transgenic animal models with perturbations of individual compounds of the GH regulatory system to study somatotrope signaling. Impaired GH signaling is present in the lit mouse, which has a GHRH receptor (GHRH-R) mutation, and the dw rat, which has a post-receptor signaling defect. Both models also have impaired response to GH secretagogues (GHS), implying an interaction between the two signaling systems. The spontaneous dwarf rat (SDR), in which a mutation of the GH gene results in total absence of the hormone, shows characteristic changes in the hypothalamic regulatory hormones due to an absence of GH feedback and alterations in the expression of each of their pituitary receptors. Treatment of SDRs with GHRH and a GHS has allowed demonstration of a stimulatory effect GHRH on GHRH-R and GHS-R, and somatostatin receptor type 2 (sst2) expression and an inhibitory effect on sst5 expression. GH also modifies the expression of these receptors, though its effects are seen at later time periods and appear to be indirect. In the absence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor stimulation of GH synthesis and release. However, in the presence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor suppression of GH synthesis and release. Loss of liver insulin-like growth factor I (IGF-I) feedback on the hypothalamic-pituitary system increases GH secretion, which, in turn, stimulates liver growth. Depletion of liver-derived IGF-I increases the expression and sensitivity of pituitary GHRH-R and GHS-R. The major site of action of liver-derived IGF-I in the regulation of GH secretion is at the pituitary level. Neuropeptide Y (NPY) is not required for basal regulation of the GH axis. NPY is required for fasting-induced suppression of GHRH and SRIH expression. NPY is also required for fasting-induced augmentation of pituitary GHS-R mRNA. Overall, the results indicate a complex regulation of GH secretion in which somatotrope receptor, as well as ligand expression, exerts an important physiological role.