Ghrelin Decreases Firing Activity of Gonadotropin-Releasing Hormone (GnRH) Neurons in an Estrous Cycle and Endocannabinoid Signaling Dependent Manner.

The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R) in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca(2+)-imaging revealed a ghrelin-triggered increase of the Ca(2+)-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10µM) suggesting direct action of ghrelin. Estradiol (1nM) eliminated the ghrelin-evoked rise of Ca(2+)-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40nM-4μM) administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1) antagonist AM251 (1µM) and the intracellularly applied DAG-lipase inhibitor THL (10µM), indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner

The Presence of Growth Hormone Secretagogue Receptor (ghrelin receptor) in Metabolic Tissues of Beef Cattle with Differences in Composition of Gain

Beef steers (n = 72) of similar age, weight (651 ± 3.1 lb), and genetic (Angus crossbred) background were used to determine the effects of growing diet composition (high‐forage vs. high‐concentrate) on the abundance of growth hormone secretagogue receptor (GHS‐R or ghrelin receptor) in metabolically important tissues of beef cattle. At trial initiation (d 0), 8 steers were harvested for initial carcass composition. The remaining 64 steers were allotted, by weight, to pen and treatment was assigned randomly. Treatments were 1) a high‐forage diet fed during the growing period (116 d) followed by a high‐concentrate diet during the finishing period (117‐209 d; GRW‐FNSH) or 2) a high‐concentrate diet fed for the duration of the trial (0‐209 d; FNSH‐FNSH). Steers were allowed ad libitum consumption regardless of dietary treatment. Eight steers per treatment were harvested on d 88, 116, 165, and 209. Immediately following harvest, liver, muscle (sternomandibularis), and subcutaneous adipose tissue samples were collected from each steer and immersed in liquid nitrogen. Longissimus dorsi samples were collected following a 48 h chill to establish a preliminary analysis of GHS‐R abundance within an economically important muscle tissue. Protein separation and quantification was determined using SDSPAGE and Western blotting techniques. Protein abundance was detected using the LI‐COR® system and standardized to β‐Actin. Protein abundance data were analyzed statistically using the GLM procedure of SAS comparing diet, harvest date, and their interaction. Protein abundance of GHS‐R in longissimus dorsi tissue fluctuated relative to serial harvest date (P \u3c 0.001), and was highest on d 88 in both treatment groups. The FNSH‐FNSH steers had increased abundance of GHS‐R in longissimus dorsi on d 88 and 116 compared with the GRW‐FNSH steers. A dietary treatment by serial harvest day interaction (P \u3c 0.05) occurred for protein abundance of GHS‐R in subcutaneous adipose tissue. Abundance of GHS‐R in subcutaneous adipose tissue of the GRW‐FNSH was greatest on d 88, whereas abundance for the FNSHFNSH treatment was greatest at the end of the finishing period (d 209). An interaction of dietary treatment and serial harvest day resulted (P \u3c 0.05) for GHS‐R abundance in liver tissue. The GRW‐FNSH steers had increased liver GHS‐R abundance following realimentation compared with the FNSH‐FNSH steers which were on a continuous plane of nutrition. Protein abundance for liver GHS‐R in both dietary treatments increased quadraticly (P \u3c 0.001) throughout the feeding period. The GHS‐R was not detected in sternomandibularis tissue. Overall liver GHS‐R abundance increased in both dietary treatments following realimentation which is inconsistent with our hypothesis. Increased GHS‐R abundance in various tissues of beef cattle while ghrelin concentrations are high and excess fat deposition is occurring warrants further investigation

Effect of Bacterial Infection on Ghrelin Receptor Regulation in Periodontal Cells and Tissues.

The effect of bacterial infection on the expression of growth hormone secretagogue receptor (GHS-R) was investigated in periodontal cells and tissues, and the actions of ghrelin were evaluated. GHS-R was assessed in periodontal tissues of rats with and without periodontitis. Human gingival fibroblasts (HGFs) were exposed to Fusobacterium nucleatum in the presence and absence of ghrelin. GHS-R expression was determined by real-time PCR and immunocytochemistry. Furthermore, wound healing, cell viability, proliferation, and migration were evaluated. GHS-R expression was significantly higher at periodontitis sites as compared to healthy sites in rat tissues. F. nucleatum significantly increased the GHS-R expression and protein level in HGFs. Moreover, ghrelin significantly abrogated the stimulatory effects of F. nucleatum on CCL2 and IL-6 expressions in HGFs and did not affect cell viability and proliferation significantly. Ghrelin stimulated while F. nucleatum decreased wound closure, probably due to reduced cell migration. Our results show original evidence that bacterial infection upregulates GHS-R in rat periodontal tissues and HGFs. Moreover, our study shows that ghrelin inhibited the proinflammatory actions of F. nucleatum on HGFs without interfering with cell viability and proliferation, suggesting that ghrelin and its receptor may act as a protective molecule during bacterial infection on periodontal cells

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.

Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr(3)(octanoyl), Lys(19)(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys(19)(Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues

Unacylated ghrelin promotes adipogenesis in rodent bone marrow via ghrelin O-acyl transferase and GHS-R1a activity: evidence for target cell-induced acylation

Despite being unable to activate the cognate ghrelin receptor (GHS-R), unacylated ghrelin (UAG) possesses a unique activity spectrum that includes promoting bone marrow adipogenesis. Since a receptor mediating this action has not been identified, we re-appraised the potential interaction of UAG with GHS-R in the regulation of bone marrow adiposity. Surprisingly, the adipogenic effects of intra-bone marrow (ibm)-infused acylated ghrelin (AG) and UAG were abolished in male GHS-R-null mice. Gas chromatography showed that isolated tibial marrow adipocytes contain the medium-chain fatty acids utilised in the acylation of UAG, including octanoic acid. Additionally, immunohistochemistry and immunogold electron microscopy revealed that tibial marrow adipocytes show prominent expression of the UAG-activating enzyme ghrelin O-acyl transferase (GOAT), which is located in the membranes of lipid trafficking vesicles and in the plasma membrane. Finally, the adipogenic effect of ibm-infused UAG was completely abolished in GOAT-KO mice. Thus, the adipogenic action of exogenous UAG in tibial marrow is dependent upon acylation by GOAT and activation of GHS-R. This suggests that UAG is subject to target cell-mediated activation – a novel mechanism for manipulating hormone activity

Ablations of Ghrelin and Ghrelin Receptor Exhibit Differential Metabolic Phenotypes and Thermogenic Capacity during Aging

mice are adaptive. mice.Our data therefore suggest that GHS-R ablation activates adaptive thermogenic function(s) in BAT and increases EE, thereby enabling the retention of a lean phenotype. This is the first direct evidence that the ghrelin signaling pathway regulates fat-burning BAT to affect energy balance during aging. This regulation is likely mediated through an as-yet-unidentified new ligand of GHS-R

Molecular ghrelin system in the pancreatic acinar cells : the role of the polypeptide, caerulein and sensory nerves

Ghrelin (GHRL) is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Experimental studies showed that GHRL protects the stomach and pancreas against acute damage, but the effect of GHRL on pancreatic acinar cells was still undetermined. Aim: To investigate the effect of GHRL and caerulein on the functional ghrelin system in pancreatic acinar cells taking into account the role of sensory nerves (SN). Methods: Experiments were carried out on isolated pancreatic acinar cells and AR42J cells. Before acinar cells isolation, GHRL was administered intraperitoneally at a dose of 50 µg/kg to rats with intact SN or with capsaicin deactivation of SN (CDSN). After isolation, pancreatic acinar cells were incubated in caerulein-free or caerulein containing solution. AR42J cells were incubated under basal conditions and stimulated with caerulein, GHRL or a combination of the above. Results: Incubation of isolated acinar cells with caerulein inhibited GHS-R and GHRL expression at the level of mRNA and protein in those cells. Either in rats with intact SN or with CDSN, administration of GHRL before isolation of acinar cells increased expression of GHRL and GHS-R in those cells and reversed the caerulein-induced reduction in expression of those parameters. Similar upregulation of GHS-R and GHRL was observed after administration of GHRL in AR42J cells. Conclusions: GHRL stimulates its own expression and expression of its receptor in isolated pancreatic acinar cells and AR42J cells on the positive feedback pathway. This mechanism seems to participate in the pancreatoprotective effect of GHRL in the course of acute pancreatitis

Contribution of Dynamic and Genetic Tests for Short Stature Diagnosing: A Case Report

Genetics; Macimorelin; Short statureGenètica; Macimorelina; Baixa estaturaGenética; Macimorelina; Baja estaturaGenetic tests have led to the discovery of many novel genetic variants related to growth failure, but the clinical significance of some results is not always easy to establish. The aim of this report is to describe both clinical phenotype and genetic characteristics in an adult patient with short stature associated with a homozygous variant in disintegrin and metalloproteinase with thrombospondin motifs type 17 gene (ADAMTS17) combined with a homozygous variant in the GH secretagogue receptor (GHS-R). The index case had severe short stature (SS) (−3.0 SD), small hands and feet, associated with eye disturbances. Genetic tests revealed homozygous compounds for ADAMTS17 responsible for Weill–Marchesani-like syndrome but a homozygous variant in GHS-R was also detected. Dynamic stimulation with an insulin tolerance test showed a normal elevation of GH, while the GH response to macimorelin stimulus was totally flattened. We show the implication of the GHS-R variant and review the molecular mechanisms of both entities. These results allowed us to better interpret the phenotypic spectrum, associated co-morbidities, its implications in dynamic tests, genetic counselling and treatment options not only to the index case but also for her relatives

Role of Ghrelin Receptor in Sarcopenia: Involvement of Redox Signaling and RANKL.

Sarcopenia is aging-induced debilitating loss of skeletal muscle strength and function. Evidence from aging research suggests an integration of disrupted mechanotransduction and oxidative stress elevation that leads to muscle atrophy. Ghrelin is a 28 amino acid peptide hormone circulating in both acylated (AG) and unacylated (UAG) forms that acts as a nutrient sensor and metabolic regulator. Acylated ghrelin then binds to the growth hormone secretagogue receptor (GHS-R) or the “ghrelin receptor.” Emerging evidence indicates that GHS-R knockout may hold greater promise in protecting against sarcopenia and fasting-induced atrophy (Wu 2020, Sun 2020). Furthermore, ghrelin can promote the elevation of nNOS, which is important as nNOS is critical to the preservation of the morphology and strength of the skeletal muscle (Lawler, 2021). Ghrelin can also stimulate protective proteins (e.g., HSP70, SIRT1), which attenuate Nox2 and ROS production. In our laboratory, recent preliminary data demonstrated positive feedback between Nox2 and RANKL that amplifies pathology and attenuates ca2+ overload, damage, and inflammation in dystrophic skeletal muscle. However, this feedback between RANKL and Nox2, and the redox regulation in sarcopenia is unknown. PURPOSE: Thus, we propose that GHS-R-dependent ghrelin pathways attenuate the markers of damage, inflammation, and redox regulation that mitigate the RANKL/Nox2 feedback and fibrosis in the aged muscles-induced sarcopenia. METHODS: Two different age C57Bl/6 mice were divided into four different groups (n = 6/group): (a) 11 months middle age wildtype, (b) Middle age WT + GHS-R -/-, (c) 23 months old age wildtype, (d) old age WT + GHS-R -/-. At the end of the experiments, gastrocnemius muscles were dissected and snap-frozen for further analysis. RESULTS: Data of protein abundance by western blotting revealed that elevated Nox2 complex marker (p67 phox), and Nox4 are decreased in the GHSR -/- groups in both middle and old-age mice. RANKL levels are significantly increased in the old group compared to the middle age. Furthermore, knocking out the GHSR mitigate the elevated RANKL levels. Markers for the damage, inflammation, Ca2+ overload, and sarcopenia-related sarcolemma-DGC dysregulation are currently being investigated. CONCLUSION: In conclusion, we demonstrate the contribution of RANKL, Nox2, and Nox4 in aging-induced sarcopenia. GHSR-dependent ghrelin pathway knockout shows a promising mechanism to mitigate oxidative stress, damage, and inflammation in sarcopenia. Further analysis will be performed to fully understand these underlying mechanisms

Vasodilator action of ghrelin

Ghrelin is a 28-amino acid peptide predominantly produced in the stomach and secreted into the circulation. Ghrelin is found in plasma and tissues in two major forms of n-octanoyl-modified at its N-terminal third serine residue and des-acyl ghrelin. The n-octanoyl group of ghrelin is essential for its growth hormone (GH)-releasing activity and appetite regulation mediated through growth hormone secretagogue receptor (GHS-R). We demonstrated that both ghrelin and des-acyl ghrelin evoke vasodilatation at remarkably low concentrations compared to acetylcholine (ACh) in phenylephrine (PE)-constricted perfused rat mesenteric vascular bed (MVB). This was abolished in endothelium-denuded preparations and in endothelium-intact preparations exposed to either a calcium-activated potassium channel (KCa) blocker or a depolarizing stimulus. While KATP channel blockade, nitric oxide synthase and cyclooxygenase inhibition had no effect, the responses were abolished in the presence of combinations of apamin and charybdotoxin, apamin and TRAM-34, and ouabain and Ba2+. The GHS-R antagonist, [D-Lys3]-GHRP-6, per se evoked vasodilatation. Inclusion of L-756867, a peptide antagonist of classical GHS-R, failed to evoke any vasodilator response or to affect vasodilatation evoked by ghrelin. Both non-peptide agonists of GHS-R, L-166446 and L-163255, demonstrated concentration-dependent decreases in perfusion pressure. All short peptides encompassing the first 20, 16, 10, 6, 4, and 3 residues of des-acyl ghrelin were able to evoke vasodilator responses to the same extent as des-acyl ghrelin. However, vasodilatation to single amino acids, L-serine and glycine, were significantly attenuated. Streptozotocin (STZ)-induced diabetes increased plasma ghrelin concentration. Diabetes for 4-weeks did not cause any significant reduction in ghrelin-evoked vasodilatation, whereas 8-weeks diabetes significantly reduced ghrelin-evoked vasodilatation. In contrast to ghrelin, there was a duration-dependent fall in vasodilator response to ACh from 4- to 8-weeks diabetes. These data suggest that the vasodilatation evoked by ghrelin is mediated by endothelium-dependent hyperpolarization (EDHF) by mechanism(s) that are independent of classical GHS-R activation. In addition, EDHF-dependent ghrelin-evoked vasodilator responses may not be affected, at least in the early stages of STZ diabetes, whereas the responses to ACh, predominantly mediated through nitric oxide, are progressively diminished right from the early stages of endothelial dysfunction in STZ diabetic rats