GH-deficient Rats Research Articles

The distinct hepatic metabolic profile and relation with impaired liver function in congenital isolated growth hormone-deficient rats.

Patients with growth hormone deficiency (GHD) with inadequate growth hormone levels are often correlated with nonalcoholic fatty liver disease (NAFLD). However, the potential mechanism of how GHD influences liver function remains obscure. In the present study, we aim to perform hepatic metabolomics in Lewis dwarf rats, which were the standard congenital isolated GH-deficient rat, to evaluate the characterizations of hepatic metabolic profiles and explore their relations with liver functions. Lewis dwarf homozygous (dw/dw) rats at 37 weeks (five females and five males), and Lewis dwarf heterozygous (dw/+) rats at 37 weeks (five females and five males) were analyzed in our study. Body lengths and weights, liver weights, serum alanine transaminase (ALT), and serum aspartate transaminase (AST) were measured. ELISA and RT-qPCR were used to assess IGF-1 levels in serum and liver, respectively. The non-targeted metabolomics was performed in the livers of dw/+ and dw/dw rats. Differential metabolites were selected according to the coefficient of variation (CV), variable importance in the projection (VIP) > 1, and P < 0.05. Hierarchical clustering of differential metabolites was conducted, and the KEGG database was used for metabolic pathway analysis. The body weights, body lengths, liver weights, and IGF-1 levels in the serum and liver of dw/dw rats were significantly decreased compared with dw/+ rats. Dw/dw rats exhibited more obvious hepatic steatosis accompanied by higher serum ALT and AST levels. Hepatic metabolomics showed that a total of 88 differential metabolites in positive ion mode, and 51 metabolites in negative ion mode were identified. Among them, lysophosphatidylcholine (LPC) 16:2, LPC 18:3, LPC 22:6, fatty acid esters of hydroxy fatty acids (FAHFA)18:1 were significantly decreased, while palmitoyl acid, dehydrocholic acid, and 7-ketolithocholic acid were significantly increased in dw/dw rats compared with dw/+ rats. These seven differential metabolites were significantly associated with phenotypes of rats. Finally, KEGG pathway analysis showed that the arginine and proline metabolism pathway and bile secretion pathway were mainly clustered. Lewis dw/dw rats with congenital isolated growth hormone deficiency (IGHD) showed liver steatosis and abnormal liver function, which could be potentially associated with the distinctive hepatic metabolic profiles.

Dexamethasone and BCAA Failed to Modulate Muscle Mass and mTOR Signaling in GH-Deficient Rats.

Branched-chain amino acids (BCAAs) and IGF-I, the secretion of which is stimulated by growth hormone (GH), prevent muscle atrophy. mTOR plays a pivotal role in the protective actions of BCAA and IGF-1. The pathway by which BCAA activates mTOR is different from that of IGF-1, which suggests that BCAA and GH work independently. We tried to examine whether BCAA exerts a protective effect against dexamethasone (Dex)-induced muscle atrophy independently of GH using GH-deficient spontaneous dwarf rats (SDRs). Unexpectedly, Dex did not induce muscle atrophy assessed by the measurement of cross-sectional area (CSA) of the muscle fibers and did not increase atrogin-1, MuRF1 and REDD1 expressions, which are activated during protein degradation. Glucocorticoid (GR) mRNA levels were higher in SDRs compared to GH-treated SDRs, indicating that the low expression of GR is not the reason of the defect of Dex’s action in SDRs. BCAA did not stimulate the phosphorylation of p70S6K or 4E-BP1, which stimulate protein synthesis. BCAA did not decrease the mRNA level of atrogin-1 or MuRF1. These findings suggested that Dex failed to modulate muscle mass and that BCAA was unable to activate mTOR in SDRs because these phosphorylations of p70S6K and 4E-BP1 and the reductions of these mRNAs are regulated by mTOR. In contrast, after GH supplementation, these responses to Dex were normalized and muscle fiber CSA was decreased by Dex. BCAA prevented the Dex-induced decrease in CSA. BCAA increased the phosphorylation of p70S6K and decreased the Dex-induced elevations of atrogin-1 and Bnip3 mRNAs. However, the amount of mTORC1 components including mTOR was not decreased in the SDRs compared to the normal rats. These findings suggest that GH increases mTORC1 activity but not its content to recover the action of BCAA in SDRs and that GH is required for actions of Dex and BCAA in muscles.

The effect and mechanism of growth hormone replacement on cognitive function in rats with traumatic brain injury.

ObjectiveThe effects of growth hormone on cognitive dysfunction were observed in a controlled cortical impact (CCI) rat model and the underlying mechanism was explored.MethodThree-month-old male SD rats were randomly divided into sham (n = 10), control (n = 10), and CCI groups (n = 40) The parameters were set as follows: striking speed, 3.5 m/s; impact depth, 1.5 mm; and dwell time, 400 msec. Eight and ten weeks post-injury, the GH levels were measured the water maze test and novel object recognition test were performed. CCI rats were divided into normal and decreased GH groups, and further randomly divided into two sub-groups (rhGH treatment and saline vehicle groups). All rats were tested for SYN, BDNF, and TrkB mRNA in the prefrontal cortex and hippocampus by RT-PCR.ResultsCCI rats 8 weeks post-injury had cognitive dysfunction regardless of the GH level (P<0.05). rhGH treatment improved cognitive function in CCI rats. There was a positive correlation between the expression of prefrontal BDNF and SYN mRNA in CCI rats after rhGH therapy and the water maze test score (r = 0.773 and 0.534, respectively; P<0.05). Furthermore, the expression of BDNF, TrkB, and SYN mRNA in the hippocampus was negatively correlated with the water maze test score (r = 0.602, 0.773, 0.672, and 0.783, respectively; P<0.05). There was a difference in the expression of hippocampal and prefrontal BDNF, TrkB, and SYN mRNA (P<0.05)ConclusionrhGH treatment had a positive effect on cognitive function, which was more evident in GH-deficient rats. The increased expression of hippocampal and prefrontal BDNF and TrkB mRNA is implicated in rhGH therapy to improve cognitive function. Changes in the expression of hippocampal SYN mRNA following rhGH therapy may also play a role in improving cognitive function.

GH-independent IGF-I action is essential to prevent the development of nonalcoholic steatohepatitis in a GH-deficient rat model

The progression to nonalcoholic steatohepatitis (NASH) from simple steatosis is associated with the mitochondrial dysfunction, enhanced oxidative stress, and inflammation. Recently, it has been reported that the prevalence of NAFLD (nonalcoholic fatty liver disease)/NASH is increased in patients with adult growth hormone deficiency (AGHD), suggesting that the deficiencies in GH and insulin-like growth factor (IGF-I) are involved in the development of NAFLD/NASH; however, the precise underlying mechanism remains to be elucidated. To clarify the mechanisms and the specific contribution of GH and IGF-I in these conditions, we examined the liver of a GH-deficient rat model, spontaneous dwarf rat (SDR) and the effect of GH and IGF-I administration. SDR showed steatosis and fibrosis in the liver in line with the phenotype observed in AGHD. Serum AST and ALT levels and triglyceride content in the liver were significantly increased in the SDR compared with the control. Intriguingly, the mitochondrial morphology in the SDR hepatocyte was impaired and the area was significantly decreased. Furthermore, oxidative stress in the SDR liver was enhanced. These changes were improved not only by GH but also by IGF-I administration, suggesting that GH-independent IGF-I action plays an essential role in the liver. In conclusion, we demonstrated that GH-deficient rat exhibits NASH and IGF-I plays an essential role to prevent the development of NASH. The improved mitochondrial function and reduced oxidative stress may contribute the effect of IGF-I in the liver.

Pharmacodynamic responses of plasma and tissue C-type natriuretic peptide to GH: correlation with linear growth in GH-deficient rats

Studies from genetic modification and spontaneous mutations show that C-type natriuretic peptide (CNP) signalling plays an essential part in postnatal endochondral growth, but measurement of CNP proteins and changes in their abundance in tissues and plasma during normal growth has not been reported. Using rodent pups with GH deficiency, we now describe the pharmacodynamic response of CNP and rat amino-terminal proCNP (NTproCNP) in plasma and tissues, and relate these to changes in linear growth (nose-tail length, tibial length and tibial growth plate width) during the course of 1 week of GH or saline (control) administration. Compared with saline, significant increases in plasma and tissue CNP forms were observed after 24 h in GH-treated pups and before any detectable change in linear growth. Whereas CNP abundance was increased in most tissues (muscle, heart and liver) by GH, enrichment was the greatest in extracts from growth plates and kidney. Plasma and tissue concentrations in GH-treated pups were sustained or further increased at 1 week when strong positive associations were found between plasma NTproCNP and linear growth or tissue concentrations. High content of NTproCNP in kidney tissue strongly correlated with plasma concentrations, which is consistent with previous data showing renal extraction of the peptide. In showing a prompt and significant increase in CNP in tissues driving normal endochondral growth, these findings provide further rationale for CNP agonists in the treatment of growth disorders resistant to current therapies and support the use of CNP concentrations as biomarkers of linear growth.

Optimized clinical performance of growth hormone with an expanded genetic code

The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.

N-Glycosylation Increases the Circulatory Half-Life of Human Growth Hormone

Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expressed in HEK293 cells. In a scan of 15 different positions for N-glycosylation sites, four positions (amino acids 93, 98, 99, and 101) were efficiently utilized and did not influence GH in vitro activity. A GH variant (3N-GH) with all these sites was produced in CHOK1SV cells and contained up to three N-glycans. Two pools of 3N-GH were purified and separated according to their charge by anion-exchange chromatography. Anion-exchange HPLC revealed that the N-glycans in the two pools were very similar except for the extent of sialylation. Both 3N-GH pools circulated longer in rats than wild-type GH. The terminal half-life of 3N-GH after iv injection was 24-fold prolonged compared with wild-type GH for the pool with the most pronounced sialylation, 13-fold prolonged for the less sialylated pool, and similar to the wild-type for desialylated 3N-GH. The less sialylated 3N-GH pool exhibited a profound pharmacodynamic effect in GH-deficient rats. Over a 4-d period, a single injection of 3N-GH induced a stronger IGF-I response and a larger increase in body weight than daily injections with wild-type GH. Thus, N-glycans can prolong the in vivo circulation and enhance the pharmacodynamic effect of GH. Sialic acids seem to play a pivotal role for the properties of glycosylated GH.

Timing effects of growth hormone supplementation on rat craniofacial growth

Growth hormone (GH) supplementation has become a popular treatment approach for GH normal children with short stature. To investigate how the timing of GH supplementation affects the growth of the craniofacial region, three groups of GH-normal, 28-day-old female Wistar rats were examined over 4 weeks: the early group (n = 10) received two daily injections of rhGH (2 mg/kg/day) from days 1 to 28, the late group (n = 10) received two daily saline injections from days 1 to 14 (Phase I) followed by two daily injections of rhGH from days 14 to 28 (Phase II), and the control group (n = 12) received two daily saline injections from days 1 to 28. Lateral cephalometric, forelimb and hindlimb radiographs obtained weekly were scanned, standardized points digitized, and distances were measured using the Viewbox software. Growth curves between groups were compared using multilevel iterative generalized least squares curve fitting procedures. Supplementation during Phase I in the early group produced significant treatment effects in cranial and cranial base, midface, posterior corpus, and limb lengths which varied inversely with relative maturity (percentage growth completed at the start of the study). During Phase II, GH supplementation in the early and late groups showed treatment effects as above and additional viscerocranial and mandibular measurements, but these effects were unrelated to the relative maturity of the variables. These latter results are at variance with earlier findings in GH-deficient rats, raising the possibility that that GH-normal rats may not respond to GH supplementation in a similar fashion to GH-deficient rats.

Mode of Growth Hormone Action in Osteoblasts

Growth hormone (GH) affects bone size and mass in part through stimulating insulin-like growth factor type 1 (IGF-1) production in liver and bone. Whether GH acts independent of IGF-1 in bone remains unclear. To define the mode of GH action in bone, we have used a Cre/loxP system in which the type 1 IGF-1 receptor (Igf1r) has been disrupted specifically in osteoblasts in vitro and in vivo. Calvarial osteoblasts from mice homozygous for the floxed IGF-1R allele (IGF-1R(flox/flox)) were infected with adenoviral vectors expressing Cre. Disruption of IGF-1R mRNA (>90%) was accompanied by near elimination of IGF-1R protein but retention of GHR protein. GH-induced STAT5 activation was consistently greater in osteoblasts with an intact IGF-1R. Osteoblasts lacking IGF-1R retained GH-induced ERK and Akt phosphorylation and GH-stimulated IGF-1 and IGFBP-3 mRNA expression. GH-induced osteoblast proliferation was abolished by Cre-mediated disruption of the IGF-1R or co-incubation of cells with an IGF-1-neutralizing antibody. By contrast, GH inhibited apoptosis in osteoblasts lacking the IGF-1R. To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in similarly treated mice lacking the IGF-1R in osteoblasts were not significantly affected. These results indicate that although direct IGF-1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo.

Growth Hormone is a Stimulating but Not An Essential Factor in Healing of Colon

Growth hormone (GH) has been implicated as an important factor in the healing and previous studies showed significant strength acceleration of experimental intestinal anastomoses. To study the healing of experimental colonic anastomoses in GH-deficient rats and to study the potential physiological effects of GH-substitution on healing parameters. Exogenous rhGH treatment started 7 days prior to surgery and continued until day 4 postoperatively accelerates the strength development of the experimental colonic anastomoses in dwarf rats indicating a potent role of growth hormone in colonic healing. However, GH is not essential in the healing process, since anastomotic healing in GH-deficient dwarf rats is like rats with normal pituitary function.

Regulation of IGF‐1 signaling by integrins in bone: Role of mechanical loading

Skeletal unloading by hindlimb suspension results in diminished bone formation associated with altered proliferation and differentiation of osteoprogenitor cells and decreased number and function of osteoblasts. This is associated with resistance to IGF-1. Therefore, alterations of the IGF-1 signaling pathway were evaluated to characterize the osteopenic changes of skeletal unloading. Twelve week old GH deficient (dw-4) and Sprague-Dawley rats were hindlimb unloaded by tail suspension for 7 and 14 days. In vivo IGF-1 administration (2.5mg/kg/day) to unloaded animals revealed unloading-induced IGF-1 resistance. Bone formation rates at the tibiofibular junction were significantly reduced by 30–40% and the stimulatory effects of IGF-1 blunted by unloading. Assessment of bone marrow osteoprogenitor (BMOp) cells from unloaded animals demonstrated significantly decreased numbers that failed to increase with in vivo IGF-1 treatment. BMOp cells from unloaded animals failed to respond to in vitro IGF-1 stimulation with respect to activation of the IGF-1 receptor and subsequent activation of the ras/MAPK/ERK and PI3K/Akt pathways. Alterations of the IGF-1 signaling pathway were not secondary to increased phosphatase activity or altered ligand-receptor binding. Inhibition of integrin signaling by the disintegrin, echistatin, recapitulated the unloading phenotype by blunting IGF-1 stimulated IGF-1 receptor phosphorylation and cell proliferation. Additionally, mRNA expression of multiple integrin subunits was reduced by 50–90% in BMOp cells from unloaded animals compared to loaded controls. We conclude that integrin signaling plays a critical role in IGF-1 signaling and contributes to the resistance to IGF-1 induced by skeletal unloading. Supported by NIH RO1 DK54793 and NASA NAG 2-1371.

Regulation of Peptide YY Levels by Age, Hormonal, and Nutritional Status

Peptide YY (PYY) 3-36 has recently been recognized as an important gut hormone that influences food intake. Peripheral injections of PYY 3-36 in rats inhibit food intake in experimental animals as well as in lean and obese human subjects. This hormone has been suggested as an attractive therapeutic option for obesity. The aim of this study was to assess the influence of age, sex, thyroid status, growth hormone (GH), pregnancy, and food restriction on PYY levels in rat. We determined plasma PYY levels in all experimental sets. PYY levels were influenced by age, with the highest hormone levels achieved in early postnatal life (day 10) and decreasing thereafter. PYY levels were also dependent on thyroid hormone status being decreased in hyperthyroid rats. Exogenous GH administration led to a clear-cut decrease in PYY levels in both normal and GH-deficient rats. Acute food deprivation or chronic food restriction led to decreased PYY levels in virgin and pregnant rats. In pregnant rats with food available ad libitum, PYY levels were enhanced at late gestation. Our observations indicate that PYY levels are influenced by age, thyroid hormones, and GH. These data indicate that PYY could be involved in the changes of food intake associated with these conditions. The PYY levels observed in acute and chronic food-restricted rats indicate that, in situations of decreased energy intake, the lower PYY levels could serve to disinhibit central pathways and facilitate food intake.

Mechanisms of Growth Hormone (GH) Action: IDENTIFICATION OF CONSERVED Stat5 BINDING SITES THAT MEDIATE GH-INDUCED INSULIN-LIKE GROWTH FACTOR-I GENE ACTIVATION

Many of the actions of growth hormone (GH) on somatic growth and tissue maintenance are mediated by insulin-like growth factor-I (IGF-I), a secreted protein whose gene expression is rapidly and potently induced by GH by unknown mechanisms. Recent studies implicating Stat5a and Stat5b in the growth response to GH in mice and observations linking Stat5b to control of IGF-I gene transcription in rats have prompted the current investigations into the molecular determinants of a putative regulatory network extending from GH through Stat5b to IGF-I. Here we characterize as critical components of this hormone-activated transcriptional pathway two adjacent Stat5 binding sites in the second intron of the rat IGF-I gene located within a conserved region previously found to undergo acute and reversible changes in chromatin structure after in vivo GH treatment. As assessed by chromatin immunoprecipitation assays, GH rapidly induced binding of Stat5 to this DNA segment in the liver of GH-deficient rats, just prior to the onset of transcription from both major and minor IGF-I gene promoters. Biochemical reconstitution experiments showed that the two intronic Stat5 DNA elements were able to bind Stat5b in vitro after GH treatment could transmit GH- and Stat5b-dependent transcriptional responsiveness to the major IGF-I promoter and to a minimal neutral gene promoter and were required for full stimulation of reporter gene activity by GH. Taken together, these results identify an intronic enhancer as a key mediator of GH-induced IGF-I gene transcription working through Stat5b and provide new insight into the molecular architecture of this transcriptional pathway.

Ghrelin improves endothelial dysfunction through growth hormone-independent mechanisms in rats

Ghrelin is a novel growth hormone (GH)-releasing peptide which was isolated from the stomach. We have reported that ghrelin causes vasorelaxation in rats through GH-independent mechanisms. We investigated whether ghrelin improves endothelial dysfunction. Ghrelin was subcutaneously administered to GH-deficient rats for three weeks. After isolation of the thoracic aorta, aortic ring tension was measured to evaluate vasorelaxation. Acetylcholine-induced vasorelaxation was impaired in GH-deficient rats given placebo compared to that in normal rats given placebo. GH-deficient rats treated with ghrelin, however, showed a significant increase in the maximal relaxation as compared with those given placebo. This improvement by ghrelin was inhibited by N G-nitro- l-arginine methyl ester, a nonselective nitric oxide synthase (NOS) inhibitor. Western blot analysis demonstrated that treatment with ghrelin increased endothelial NOS (eNOS) expression in the aorta of GH-deficient rats. These results suggest that administration of ghrelin improves endothelial dysfunction and increases eNOS expression in rats through GH-independent mechanisms.

Reduced hypothalamic neuropeptide Y expression in growth hormone- and prolactin-deficient Ames and Snell dwarf mice.

Neuropeptide Y (NPY)-producing neurons in the hypothalamic arcuate nucleus (ARC) have been implicated in GH feedback in several studies in rats. Ames (df/df) and Snell (dw/dw) dwarf mice carry mutations in transcription factors Prop-1 and Pit-1, respectively, that abrogate detectable expression of GH, prolactin, and TSH. The present study was undertaken to determine whether and to what extent hypothalamic NPY neurons are affected by the lifelong absence of pituitary hormone feedback in hypopituitary Ames and Snell dwarf mice. Total ARC NPY mRNA levels were quantified using in situ hybridization, and numbers of ARC NPY-producing cells were quantified using immunocytochemistry. For in situ hybridization, dwarf and normal coronal brain sections were hybridized with 35S-labeled riboprobe complementary to rat NPY cDNA, and then analyzed for total signal intensity from the entire ARC for each animal as well as for mRNA per neuron. NPY-containing perikarya in ARC were counted in sections of colchicine-treated (intracerebroventricular) dwarf and normal mice. Total ARC NPY mRNA was reduced in df/df mice to 33.6% (P < 0.01) of that in normal littermates, and reduced in dw/dw mice to 46.3% (P < 0.05) of normals, but there was no difference in expression per neuron as determined by reduced silver-grain counting. The decrement in dwarf mice of total ARC NPY mRNA without reduction in mRNA per cell suggested a reduction in NPY-containing neuron number, which was the case as shown by immunocytochemistry. NPY neuronal number in adult Ames dwarf mice (1048 +/- 104) was significantly (P < 0.01) reduced to 68% of that in DF/df littermates (1536 +/- 73), and significantly (P < 0.05) reduced in Snell dwarf mice to 63% of normals (1138 +/- 137 vs. 1726 +/- 205). This study represents the first enumeration of NPY-producing neurons in mouse hypothalamus and the first demonstration of lower NPY neuron number in a hypopituitary model. The reduction in total NPY mRNA was greater than that reported in studies of GH-deficient rats, suggesting that NPY expression may be affected by the lifelong absence of prolactin or TSH or both, as well as GH.

Cellular distribution and regulation of ghrelin messenger ribonucleic acid in the rat pituitary gland.

Ghrelin, a 28-amino-acid acylated peptide, strongly stimulates GH release and food intake. In the present study, we found that ghrelin is expressed in somatotrophs, lactotrophs, and thyrotrophs but not in corticotrophs or gonadotrophs of rat pituitary. Persistent expression of the ghrelin gene is found during postnatal development in male and female rats, although the levels significantly decrease in both cases from pituitaries of 20-d-old rats onward, but at 60 d old, the levels were higher in male than female rats. This sexually dimorphic pattern appears to be mediated by estrogens because ovariectomy, but not orchidectomy, increases pituitary ghrelin mRNA levels. Taking into account that somatotroph cell function is markedly influenced by thyroid hormones, glucocorticoids, GH, and metabolic status, we also assessed such influence. We found that ghrelin mRNA levels decrease in hypothyroid- and glucocorticoid-treated rats, increase in GH-deficient rats (dwarf rats), and remain unaffected by food deprivation. In conclusion, we have defined the specific cell types that express ghrelin in the rat anterior pituitary gland. These data provide direct morphological evidence that ghrelin may well be acting in a paracrine-like fashion in the regulation of anterior pituitary cell function. In addition, we clearly demonstrate that pituitary ghrelin mRNA levels are age and gender dependent. Finally, we show that pituitary ghrelin mRNA levels are influenced by alteration on thyroid hormone, glucocorticoids, and GH levels but not by fasting, which indicates that the regulation of ghrelin gene expression is tissue specific.

Pattern-dependent suppression of growth hormone (GH) pulsatility by ghrelin and GH-releasing peptide-6 in moderately GH-deficient rats.

The peptide hormone ghrelin binds to the GH secretagogue receptor (GHS-R), stimulates GH secretion, and promotes adipogenesis. However, continuous GHS infusion does not stimulate skeletal growth and is associated with desensitization to further GH secretagogue treatment. In this study, 7-d intermittent (i.e. every 3 h) infusion of ghrelin, or the GH secretagogue, GH-releasing peptide-6, in the moderately GH- deficient transgenic growth-retarded rat, augmented GH secretion, leading to a sustained acceleration in skeletal growth. In contrast, continuous infusion of ghrelin, or GH-releasing peptide-6, suppressed the amplitude of spontaneous GH secretory episodes and produced only a transient increase in body weight gain. The reduction in GH secretion seen with continuous GHS-R activation was not associated with a desensitization of the pituitary to GH-releasing factor or to down-regulation of hypothalamic GHS-R mRNA expression. Continuous ghrelin treatment elicited an increase in somatostatin mRNA expression in the periventricular nuclei. Thus, exposure to continuously elevated circulating ghrelin may be responsible for the suppression of GH secretion reported in rats after prolonged starvation.

Agouti-related peptide, neuropeptide Y, and somatostatin-producing neurons are targets for ghrelin actions in the rat hypothalamus.

Ghrelin, the endogenous ligand of the GH secretagogue receptor, acts at central level to elicit GH release and regulate food intake. To elucidate the neural circuit that exerts its effects, we measured the expression of hypothalamic neuropeptides involved in weight regulation and GH secretion after ghrelin administration. Adult male rats, fed or fasted for 72 h, were treated centrally (intracerebroventicularly) with a single dose of ghrelin (5 micro g). After 2, 4, and 6 or 8 h, agouti-related peptide, melanin-concentrating hormone, neuropeptide Y, prepro-orexin, GHRH, and somatostatin mRNA levels were measured by in situ hybridization. We found that ghrelin increased agouti-related peptide and neuropeptide Y expression in the arcuate nucleus of the hypothalamus of fed and fasted rats. In contrast, no change was demonstrated in the mRNA levels of the other neuropeptides studied at any time evaluated. Finally, we examined the effect of ghrelin on GHRH and somatostatin mRNA levels in GH-deficient (dwarf) rats. Our results show that ghrelin increases somatostatin mRNA levels in the hypothalamus of these rats. This study furthers our understanding of the molecular basis and mechanisms involved in the effect of ghrelin on food intake and GH secretion.

Cellular Location and Hormonal Regulation of Ghrelin Expression in Rat Testis1

Ghrelin, the endogenous ligand for the growth hormone-secretagogue receptor, is a recently cloned 28-amino acid peptide, expressed primarily in the stomach and hypothalamus, with the ability to stimulate growth hormone (GH) release and food intake. However, the possibility of additional, as yet unknown biological actions of ghrelin has been suggested. As a continuation of our recent findings on the expression and functional role of ghrelin in rat testis, we report here the pattern of cellular expression of ghrelin peptide in rat testis during postnatal development and after selective Leydig cell elimination, and we assess hormonal regulation of testicular ghrelin expression, at the mRNA and/or protein levels, in different experimental models. Immunohistochemical analyses along postnatal development demonstrated selective location of ghrelin peptide within rat testis in mature fetal- and adult-type Leydig cells. In good agreement, ghrelin protein appeared undetectable in testicular interstitium after selective Leydig cell withdrawal. In terms of hormonal regulation, testicular ghrelin mRNA and protein expression decreased to negligible levels after long-term hypophysectomy, whereas replacement with human chorionic gonadotropin (CG) (as superagonist of LH) partially restored ghrelin mRNA and peptide expression. Furthermore, acute administration of human CG (25 IU) to intact rats resulted in a transient increase in testicular ghrelin mRNA levels, with peak values 4 h after injection, an effect that was not mimicked by FSH (12.5 IU/rat). In contrast, testicular expression of ghrelin mRNA remained unaltered in GH-deficient rats, under hyper- and hypothyroidism conditions, as well as in adrenalectomized animals. In conclusion, our results demonstrate that mature Leydig cells are the source of ghrelin expression in rat testis, the protein being expressed in both fetal- and adult-type Leydig cells. In addition, our data indicate that testicular expression of ghrelin is hormonally regulated and is at least partially dependent on pituitary LH.

Growth and maturational changes in dense fibrous connective tissue following 14 days of rhGH supplementation in the dwarf rat.

The purpose of this study was to investigate the impact of recombinant human growth hormone (rhGH) on patella tendon (PT), medial collateral ligament (MCL), and lateral collateral ligament (LCL) on collagen growth and maturational changes in dwarf GH-deficient rats. Twenty male Lewis mutant dwarf rats, 37 days of age, were randomly assigned to Dwarf + rhGH (n = 10) and Dwarf + vehicle (n = 10) groups. The GH group received 1.25 mg rhGH/kg body wt twice daily for 14 days. rhGH administration stimulated dense fibrous connective tissue growth, as demonstrated by significant increases in hydroxyproline specific activity and significant decreases in the non-reducible hydroxylysylpyridinoline (HP) collagen cross-link contents. The increase in the accumulation of newly accreted collagen was 114, 67, and 117% for PT, MCL, and LCL, respectively, in 72 h. These findings suggest that a short course rhGH treatment can affect the rate of new collagen production. However, the maturation of the tendon and ligament tissues decreased 18-25% during the rapid accumulation of de novo collagen. We conclude that acute rhGH administration in a dwarf rat can up-regulate new collagen accretion in dense fibrous connective tissues, while causing a reduction in collagen maturation.