Growth Hormone-releasing Hormone Knockout Research Articles

Effects of GHRH Deficiency and GHRH Antagonism on Emotional Disorders in Mice.

Growth hormone (GH)-releasing hormone (GHRH) has been suggested to play a crucial role in brain function. We aimed to further investigate the effects of a novel GHRH antagonist of the Miami (MIA) series, MIA-602, on emotional disorders and explore the relationships between the endocrine system and mood disorders. In this context, the effects induced by MIA-602 were also analyzed in comparison to vehicle-treated mice with GH deficiency due to generalized ablation of the GHRH gene (GHRH knock out (GHRHKO)). We show that the chronic subcutaneous administration of MIA-602 to wild type (+/+) mice, as well as generalized ablation of the GHRH gene, is associated with anxiolytic and antidepressant behavior. Moreover, immunohistochemical and Western blot analyses suggested an evident activation of Nrf2, HO1, and NQO1 in the prefrontal cortex of both +/+ mice treated with MIA-602 (+/+ MIA-602) and homozygous GHRHKO (-/- control) animals. Finally, we also found significantly decreased COX-2, iNOS, NFkB, and TNF-α gene expressions, as well as increased P-AKT and AKT levels in +/+ MIA-602 and -/- control animals compared to +/+ mice treated with vehicle (+/+ control). We hypothesize that the generalized ablation of the GHRH gene leads to a dysregulation of neural pathways, which is mimicked by GHRH antagonist treatment.

Distinct physiological characteristics and altered glucagon signaling in GHRH knockout mice: Implications for longevity.

Our previous research has demonstrated that mice lacking functional growth hormone-releasing hormone (GHRH) exhibit distinct physiological characteristics, including an extended lifespan, a preference for lipid utilization during rest, mild hypoglycemia, and heightened insulin sensitivity. They also show a further increase in lifespan when subjected to caloric restriction. These findings suggest a unique response to fasting, which motivated our current study on the response to glucagon, a key hormone released from the pancreas during fasting that regulates glucose levels, energy expenditure, and metabolism. Our study investigated the effects of an acute glucagon challenge on female GHRH knockout mice and revealed that they exhibit reduced glucose production, likely due to suppressed gluconeogenesis. However, these mice showed an increase in energy expenditure. We also observed alterations in pancreatic islet architecture, with smaller islets and a reduction of insulin-producing beta cells but no changes in glucagon-producing alpha cells. Additionally, the analysis of hepatic glucagon signaling showed a decrease in glucagon receptor expression and phosphorylated CREB. In conclusion, our findings suggest that the unique metabolic phenotype observed in these long-lived mice may be partly explained by changes in glucagon signaling. Further exploration of this pathway may lead to new insights into the regulation of longevity in mammals.

L-Serine Influences Epigenetic Modifications to Improve Cognition and Behaviors in Growth Hormone-Releasing Hormone Knockout Mice.

Neurodegenerative diseases feature changes in cognition, and anxiety-like and autism-like behaviors, which are associated with epigenetic alterations such as DNA methylation and histone modifications. The amino acid L-serine has been shown to have beneficial effects on neurological symptoms. Here, we found that growth hormone-releasing hormone knockout (GHRH-KO) mice, a GH-deficiency mouse model characterized by extended lifespan and enhanced insulin sensitivity, showed a lower anxiety symptom and impairment of short-term object recognition memory and autism-like behaviors. Interestingly, L-serine administration exerted anxiolytic effects in mice and ameliorated the behavioral deficits in GHRH-KO. L-serine treatment upregulated histone epigenetic markers of H3K4me, H3K9ac, H3K14ac and H3K18ac in the hippocampus and H3K4me in the cerebral cortex in both GHRH-KO mice and wild type controls. L-serine-modulated epigenetic marker changes, in turn, were found to regulate mRNA expression of BDNF, grm3, foxp1, shank3, auts2 and marcksl1, which are involved in anxiety-, cognitive- and autism-like behaviors. Our study provides a novel insight into the beneficial effects of L-serine intervention on neuropsychological impairments.

SUN-117 Growth Hormone-Releasing Hormone (GHRH) Deficiency Promotes Inflammation Associated Carcinogenesis

The somatotropic axis, in addition to its well-known metabolic and endocrine effects, plays a pivotal role in modulation of inflammation. Additionally, GH-releasing hormone (GHRH) has been involved in the regulation of the growth of various human tumors. In this work, we aimed to investigate the consequences of GHRH and GH deficiency on the development of inflammation-associated colon carcinogenesis in a mouse model of isolated GH deficiency due to generalized ablation of the GHRH gene [GHRH knock out (GHRHKO)]. Homozygous GHRHKO (-/-) male mice and wild type (C57/BL6, +/+) male mice as control group were used.After azoxymetane (AOM)/dextran sodium sulfate (DSS) treatment GHRHKO-/- mice displayed higher Disease Activity Index (DAI) score, and more marked weight loss compared to +/+ animals. Additionally, -/- mice showed a significant increase in total colonic tumors, in particular of large size and predominantly localized in distal colon. In colonic tissue of AOM/DSS-treated -/- mice we found the presence of invasive adenocarcinomas, dysplasia and colitis with mucosal ulceration. Conversely, AOM/DSS-treated +/+ mice showed only presence of adenomas, without invasion of sub-mucosa. Treatment with AOM/DSS significantly increased prostaglandin (PG)E2 and 8-iso-PGF2α levels along with cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, nuclear factor kappa B (NFkB) and inducible nitric oxide synthase (iNOS) gene expression, in colon specimens of both groups. However, the degree of increase of all these parameters was more marked in -/- than +/+ mice.In conclusion, generalized GHRH ablation increases inflammatory response and colon carcinogenesis in male mice. Whether this results from lack of GH or GHRH remains to be established.

Transcriptomic and metabolomic profiling of long-lived growth hormone releasing hormone knock-out mice: evidence for altered mitochondrial function and amino acid metabolism.

Numerous genetic manipulations that extend lifespan in mice have been discovered over the past two decades, the most robust of which has arguably been the down regulation of growth hormone (GH) signaling. However, while decreased GH signaling has been associated with improved health and lifespan, many of the underlying physiological changes and molecular mechanisms associated with GH signaling have yet to be elucidated. To this end, we have completed the first transcriptomic and metabolomic study on long-lived growth hormone releasing hormone knockout (GHRH-KO) and wild-type mice in brown adipose tissue (transcriptomics) and blood serum (metabolomics). We find that GHRH-KO mice have increased transcript levels of mitochondrial and amino acid genes with decreased levels of extracellular matrix genes. Concurrently, mitochondrial metabolites are differentially regulated in GHRH-KO. Furthermore, we find a strong signal of genotype-by-sex interactions, suggesting the sexes have differing physiological responses to GH deficiency. Overall, our results point towards a strong influence of mitochondrial metabolism in GHRH-KO mice which potentially is tightly intertwined with their extended lifespan phenotype.

Growth hormone-releasing hormone (GHRH) deficiency promotes inflammation-associated carcinogenesis

The somatotropic axis, in addition to its well-known metabolic and endocrine effects, plays a pivotal role in modulation of inflammation. Moreover, growth hormone (GH)-releasing hormone (GHRH) has been involved in the development of various human tumors. In this work we aimed to investigate the consequences of GHRH deficiency on the development of inflammation-associated colon carcinogenesis in a mouse model of isolated GH deficiency due to generalized ablation of the GHRH gene [GHRH knock out (GHRHKO)]. Homozygous GHRHKO (-/-) male mice and wild type (C57/BL6, +/+) male mice as control group, were used.After azoxymetane (AOM)/dextran sodium sulfate (DSS) treatment -/- mice displayed higher Disease Activity Index (DAI) score, and more marked weight loss compared to +/+ animals. Additionally, -/- mice showed a significant increase in total tumors, in particular of large size predominantly localized in distal colon. In colonic tissue of AOM/DSS-treated -/- mice we found the presence of invasive adenocarcinomas, dysplasia and colitis with mucosal ulceration. Conversely, AOM/DSS-treated +/+ mice showed only presence of adenomas, without invasion of sub-mucosa.Treatment with AOM/DSS significantly increased prostaglandin (PG)E2 and 8-iso-PGF2α levels along with cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, nuclear factor kappa B (NF-kB) and inducible nitric oxide synthase (iNOS) gene expression, in colon specimens. The degree of increase of all these parameters was more markedly in -/- than +/+ mice.In conclusion, generalized GHRH ablation increases colon carcinogenesis responsiveness in male mice. Whether this results from lack of GH or GHRH remains to be established.

Increased pain and inflammatory sensitivity in growth hormone-releasing hormone (GHRH) knockout mice

Growth hormone (GH) and GH-releasing hormone (GHRH), in addition to metabolic and endocrine effects, play a role in the modulation of pain and inflammation. We aimed to elucidate the consequences of GHRH deficiency on acute nociceptive stimulation and on both acute and chronic inflammatory stimuli in a mouse model of GH deficiency. Mice with generalized ablation of the GHRH gene (GHRH knock out, GHRHKO, -/-) were compared to wild type (GHRH +/+) mice.Responsiveness to acute nociceptive stimulation and to acute inflammatory stimulation was evaluated by conventional hot plate apparatus and formalin test, respectively. We also evaluated responsiveness to colonic inflammation induced both in vivo, after dextran sodium sulfate (DSS) treatment, or ex vivo, by incubating colon segments with bacterial lipopolysaccaride (LPS). Macroscopical and histological examinations were performed, prostaglandin (PG) E2 and 8-iso-PGF2α levels and cyclooxigenase (COX)-2 and tumor necrosis factor (TNF)-α gene expression were measured.Compared to controls, -/- mice showed decreased response latency during the hot plate test, and increased licking/biting time in formalin test, particularly in the second phase of inflammation. DSS treated -/- mice showed a significant increase of colonic inflammation compared to controls. Moreover DSS treatment increased PGE2 and 8-iso-PGF2α levels, along with COX-2 and TNF-α gene expression more markedly in colon specimens of -/- mice compared to controls. LPS-induced PGE2 and 8-iso-PGF2α production from colonic segments incubated ex vivo was also increased in -/- mice.Generalized GHRH gene ablation increases sensitivity to thermal pain and both acute and persistent inflammatory stimuli in male mice.

MON-478 Antinflammatory and Antioxidant Effects of MIA-690 and MR-409 in GHRHKO Mice Colon and Prefrontal Cortex

GH-releasing hormone (GHRH) exerts various effects on multiple peripheral organs, including brain, heart, kidney and retina, in addition to its growth hormone (GH)-stimulatory effect on the pituitary somatotroph cells. Various agonistic and antagonistic analogs of GHRH have been synthesized and studied for their biological activity. In the present study we investigated the potential anti-inflammatory and antioxidant effects of the GHRH agonist, MR-409, and GHRH antagonist, MIA-690, on isolated mouse prefrontal cortex and colon specimens treated with lipopolysaccaride (LPS), a validated ex vivo model of inflammation. Specifically, we studied the effects of these two compounds on LPS-induced production of prostaglandin (PG)E2, a marker of inflammation, and 8-iso-prostaglandin (8-iso-PG)F2α, a marker of oxidative stress, both in GH deficient mice with generalized ablation of the GHRH gene (GHRH knock out, GHRHKO) (-/-) and wild type (WT, controls) (+/+) adult mice. Animals (n=8) were sacrificed and cortex and colon specimens were immediately collected and maintained in a humidified incubator with 5% CO2 at 37°C for 4 h, in RPMI buffer with added bacterial LPS (10 μg/mL) (incubation period). During the incubation period, tissues were treated with vehicle or increasing concentrations of MIA-690 (1-5 μM) and MR-409 (1-5 μM). Tissue perfusates were collected and PGE2 and 8-iso-PGF2α levels (ng/mg wet tissue) were measured by radioimmunoassay (RIA). Differences between groups were analyzed by one-way Anova followed by Newman-Keul’s. P < 0.05 was considered statistically significant. Compared to controls, -/- mice showed increased PGE2 and 8-iso-PGF2α levels after LPS-challenge in both tissues. MIA-690 and MR-409 were found to inhibit LPS-induced cortex and colon production of PGE2 and 8-iso-PGF2α in a dose-dependent manner in both +/+ and -/- mice. MIA-690 and MR-409 were significantly more effective in -/- mice compared to +/+ animals. In addition, MR-409 was more effective than MIA-690 to inhibit LPS-induced cortex and colon production of PGE2 and 8-iso-PGF2α. In conclusion, generalized GHRH ablation is associated with increased prefrontal cortex and colonic sensitivity to inflammatory stimuli, as shown by higher PGE2 and 8-iso-PGF2α production. Surprisingly, both MR-409 and MIA-690 exhibit anti-inflammatory and antioxidant effects.

Effects of growth hormone-releasing hormone gene targeted ablation on ghrelin-induced feeding

Impairment of growth hormone (GH) signaling has been associated with increased feeding and adiposity. The gastric hormone ghrelin, in addition to its GH-secretagogue effects, stimulates food intake after both central and peripheral administration. In the present study we further investigated the feeding regulatory role of the ghrelin-GH axis in a mouse model of isolated GH deficiency due to targeted ablation of the GH-releasing hormone (GHRH) gene [GHRH knockout (GHRHKO)]. We evaluated the effects of intracerebroventricular ghrelin administration on feeding behavior, related hypothalamic neuropeptides and neurotransmitters, and serum ghrelin levels in mice homozygous for GHRHKO allele (−/−) and heterozygous (+/−) control animals. Vehicle-treated GHRHKO mice showed increased food intake compared to heterozygotes, associated with increased circulating ghrelin levels. Moreover, −/− mice showed elevated hypothalamic levels of neuropeptide Y (NPY), agouti-related peptide (AgRP) mRNAs and norepinephrine (NE) and decreased corticotropin-releasing hormone (CRH) mRNA levels. Ghrelin treatment significantly augmented food intake in both genotypes, but the relative increase compared to vehicle-treated animals was higher in −/− than +/− mice. In the hypothalamus, ghrelin increased AgRP and decreased CRH gene expression only in heterozygous mice, while it induced a significant reduction in proopiomelanocortin (POMC) mRNA levels in −/− mice. Ghrelin treatment also decreased hypothalamic serotonin (5-hydroxytriptamine, 5-HT) and dopamine (DA) levels in both genotypes. Additionally, we observed increased DA metabolism induced by ghrelin in both genotypes. In conclusion, dysregulation of the ghrelin-GHRH-GH axis in GHRHKO mice could lead to increased feeding secondary to elevated circulating levels of ghrelin, and the obesogenic phenotype is likely mediated by elevated NPY and AgRP, and decreased CRH gene expression in the hypothalamus.

Increased locomotor and thermogenic activity in mice with targeted ablation of the GHRH gene

ObjectiveGrowth hormone (GH) deficiency (GHD) leads to growth failure and changes in body composition, including increased fat accumulation and reduced lean body mass in both humans and rodents. The aim of this study was to examine the factors that contribute to energy imbalance in the GH releasing hormone knock out (GHRHKO) mice, a well established model of GHD. DesignWe evaluated food intake (of standard laboratory chow), total body weight (TBW), locomotor activity, body temperature and interscapular brown adipose tissue (BAT) weight in 8 adult male mice homozygous for the GHRHKO allele (−/−) and 8 heterozygous (+/−) animals as controls. The gene expression of uncoupling protein-1 (UCP-1) in BAT and the levels of norepinephrine (NE), dopamine (DA), and serotonin (5-hydroxytryptamine, 5-HT) in the ventral striatum were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and high performance liquid chromatography (HPLC) analysis, respectively. ResultsThroughout 2months of observation −/− mice consumed approximately 40% more food (normalized to TBW; P<0.001), and showed increased locomotor activity in 24h time compared to controls (P<0.05). Moreover, −/− animals showed increased body temperature (P<0.001), BAT weight (P<0.001), and UCP-1 gene expression (P<0.001), while NE levels in the striatum area were lower (P<0.05) than controls. ConclusionsThe present study demonstrates that the increased food intake observed in GHRH ablated animals is associated with increased locomotor and thermogenic activity.

Behavioural phenotyping of male growth hormone-releasing hormone (GHRH) knockout mice

ObjectiveGH-releasing hormone (GHRH) is a key regulator of GH secretion. The role of GH in anxiety is somewhat contradictory. The aim of this study is to elucidate the consequences of lack of GHRH on emotional behaviour in a mouse model of GH deficiency due to removal of the GHRH gene (GHRH knock out, GHRHKO). DesignHomozygous GHRHKO and wild type male mice were utilized for this study. The emotional behaviour was measured through a battery of behavioural tests (locomotor activity/open field, light–dark exploration, elevated plus maze, forced swim test, tail suspension test). To correlate the emotional behaviour with brain neurochemistry, we evaluated thyrotropin-releasing hormone (TRH) gene expression in hypothalamic tissue by real-time PCR, and the levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) in prefrontal cortex by HPLC analysis. ResultsGHRHKO mice showed increased exploratory activity. In the open field test (P<0.005), light–dark box (P<0.005) and elevated plus maze (P<0.05), GHRHKO mice demonstrated a decrease in anxiety-related behaviour. In addition, GHRHKO mice showed reduced immobility time with respect to control in forced swim test and tail suspension test (P<0.0001). The gene expression of hypothalamic TRH (P<0.05) was increased, while NE levels in prefrontal cortex were decreased compared to control (P<0.05). ConclusionThese results suggest that in male mice GHRH deficiency brings about an increased physical activity and decreased anxiety- and depression-related behaviour, possibly related to increased TRH and decreased NE levels in the brain.

Increased relative amount of visceral and subcutaneous adipose tissue in long-lived GH-releasing hormone knockout (GHRHKO) mice; is it beneficial or not?

Increased relative amount of visceral and subcutaneous adipose tissue in long-lived GH-releasing hormone knockout (GHRHKO) mice; is it beneficial or not?

Growth hormone-releasing hormone disruption extends lifespan and regulates response to caloric restriction in mice

We examine the impact of targeted disruption of growth hormone-releasing hormone (GHRH) in mice on longevity and the putative mechanisms of delayed aging. GHRH knockout mice are remarkably long-lived, exhibiting major shifts in the expression of genes related to xenobiotic detoxification, stress resistance, and insulin signaling. These mutant mice also have increased adiponectin levels and alterations in glucose homeostasis consistent with the removal of the counter-insulin effects of growth hormone. While these effects overlap with those of caloric restriction, we show that the effects of caloric restriction (CR) and the GHRH mutation are additive, with lifespan of GHRH-KO mutants further increased by CR. We conclude that GHRH-KO mice feature perturbations in a network of signaling pathways related to stress resistance, metabolic control and inflammation, and therefore provide a new model that can be used to explore links between GHRH repression, downregulation of the somatotropic axis, and extended longevity. DOI:http://dx.doi.org/10.7554/eLife.01098.001.

Effects of isolated GH deficiency on adipose tissue, feeding and adipokines in mice

ObjectiveGrowth hormone deficiency (GHD) leads to growth failure and changes in body composition including increased fat accumulation and reduced lean body mass in both humans and rodents. The aim of this study was to characterize the consequences of isolated GHD (IGHD) on adiposity, total body weight (TBW), and food intake in a mouse model of autosomal recessive IGHD due to targeted ablation of the GH-releasing hormone (GHRH) gene [GHRH knockout (GHRHKO)]. Animals were also analyzed with respect to leptin, adiponectin and visfatin circulating levels and gene expression in both intra-abdominal and subcutaneous fat. DesignWe studied 8 male mice homozygous for GHRHKO allele (−/−) and 8 heterozygous (+/−) animals as controls. Feeding and TBW data were collected weekly from 3 through 5months of age. Animals were then euthanized for measurement of body length and intra-abdominal (epididymal and retroperitoneal) and subcutaneous (interscapular, axillary, gluteal and inguinal) fat weights, and for blood collection for leptin, adiponectin and visfatin measurement. Gene expression of leptin, adiponectin and visfatin in adipose tissue was evaluated by real-time reverse transcription polymerase chain reaction. ResultsGHRHKO mice had significantly increased relative intra-abdominal (P<0.01) and subcutaneous (P<0.0001) fat, accompanied by significantly increased food intake per TBW (P<0.01), whereas – despite 40% higher food consumption – TBW change was not different from controls over the 2month period. Adiponectin and visfatin mRNA levels were decreased in both intra-abdominal (P<0.001) and subcutaneous fat (P<0.0001), while leptin mRNA levels were not different from controls. Conversely, serum adiponectin levels were higher in GHRHKO mice (P<0.0001), whereas serum visfatin and leptin did not significantly differ from controls. ConclusionsIGHD due to targeted ablation of the GHRH gene in mice is associated with increased relative subcutaneous and intra-abdominal fat mass and higher food consumption which is not related to changes in circulating leptin.

Once-daily administration of CJC-1295, a long-acting growth hormone-releasing hormone (GHRH) analog, normalizes growth in the GHRH knockout mouse

Although the majority of children with isolated growth hormone (GH) deficiency have a good growth response to GH-releasing hormone (GHRH), the use of this therapeutic agent is limited by its very short half-life. Indeed, we have shown that, in mice with GHRH gene ablation (GHRH knockout; GHRHKO), even twice-daily injections of a GHRH analog are unable to normalize growth. CJC-1295 is a synthetic GHRH analog that selectively and covalently binds to endogenous albumin after injection, thereby extending its half-life and duration of action. We report the effects of CJC-1295 administration in GHRHKO animals. Three groups of 1-wk-old GHRHKO mice were treated for 5 wk with 2 microg of CJC-1295 at intervals of 24, 48, and 72 h. Placebo-treated GHRHKO mice and mice heterozygous for the GHRHKO allele served as controls. GHRHKO animals receiving daily doses of CJC-1295 exhibited normal body weight and length. Mice treated every 48 and 72 h reached higher body weight and length than placebo-treated animals, without full growth normalization. Femur and tibia length remained normal in animals treated every 24 and 48 h. Relative lean mass and subcutaneous fat mass were normal in all treated groups. CJC-1295 caused an increase in total pituitary RNA and GH mRNA, suggesting that proliferation of somatotroph cells had occurred, as confirmed by immunohistochemistry images. These findings demonstrate that treatment with once-daily administration of CJC-1295 is able to maintain normal body composition and growth in GHRHKO mice. The same dose is less effective when administered every 48 or 72 h.

Effects of long-term treatment with growth hormone-releasing peptide-2 in the GHRH knockout mouse

Growth hormone (GH) secretagogues (GHS) stimulate GH secretion in vivo in humans and in animals. They act on the ghrelin receptor, expressed in both the hypothalamus and the pituitary. It is unknown whether GHSs act predominantly by increasing the release of hypothalamic GH-releasing hormone (GHRH) or by acting directly on the somatotroph cells. We studied whether a potent GHS could stimulate growth in the absence of endogenous GHRH. To this end, we used GHRH knockout (GHRH-KO) mice. These animals have proportionate dwarfism due to severe GH deficiency (GHD) and pituitary hypoplasia due to reduced somatotroph cell mass. We treated male GHRH-KO mice for 6 wk (from week 1 to week 7 of age) with GH-releasing peptide-2 (GHRP-2, 10 microg s.c. twice a day). Chronic treatment with GHRP-2 failed to stimulate somatotroph cell proliferation and GH secretion and to promote longitudinal growth. GHRP-2-treated mice showed an increase in total body weight compared with placebo-treated animals, due to worsening of the body composition alterations typical of GHD animals. These data demonstrate that GHRP-2 failed to reverse the severe GHD caused by lack of GHRH.