Activation Of Ghrelin Receptor Research Articles

Anamorelin for cancer cachexia.

Cancer anorexia-cachexia syndrome is a multifactorial condition characterized by significant weight loss due to muscle loss. It is associated with functional impairment, changes in body composition and nutritional disorders. Ghrelin receptors are involved in the release of growth hormone (GH) in the pituitary gland and increase appetite via the hypothalamus. The secretion of GH from the pituitary gland stimulates the liver to secrete insulin-like growth factor 1 (IGF-1), which promotes muscle protein synthesis. Anamorelin is a ghrelin receptor agonist used to treat cancer cachexia. It promotes GH secretion via ghrelin receptor activation and increases appetite, resulting in increased muscle mass and weight. Clinical trials of anamorelin have demonstrated a significant increase in lean body mass index, improved cachexia and no significant increase in serious adverse events. The present review describes the processes leading to the approval of anamorelin in Japan, focusing on pharmacology, metabolism, efficacy, safety and clinical trials.

Complexes of Ghrelin GHS-R1a, GHS-R1b, and Dopamine D1 Receptors Localized in the Ventral Tegmental Area as Main Mediators of the Dopaminergic Effects of Ghrelin.

Ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R1a), is coexpressed with its truncated isoform GHS-R1b, which does not bind ghrelin or signal, but oligomerizes with GHS-R1a, exerting a complex modulatory role that depends on its relative expression. D1 dopamine receptor (D1R) and D5R constitute the two D1-like receptor subtypes. Previous studies showed that GHS-R1b also facilitates oligomerization of GHS-R1a with D1R, conferring GHS-R1a distinctive pharmacological properties. Those include a switch in the preferred coupling of GHS-R1a from Gq to Gs and the ability of D1R/D5R agonists and antagonists to counteract GHS-R1a signaling. Activation of ghrelin receptors localized in the ventral tegmental area (VTA) seems to play a significant role in the contribution of ghrelin to motivated behavior. In view of the evidence indicating that dopaminergic cells of the VTA express ghrelin receptors and D5R, but not D1R, we investigated the possible existence of functional GHS-R1a:GHS-R1b:D5R oligomeric complexes in the VTA. GHS-R1a:GHS-R1b:D5R oligomers were first demonstrated in mammalian transfected cells, and their pharmacological properties were found to be different from those of GHS-R1a:GHS-R1b:D1R oligomers, including weak Gs coupling and the ability of D1R/D5R antagonists, but not agonists, to counteract the effects of ghrelin. However, analyzing the effect of ghrelin in the rodent VTA on MAPK activation with ex vivo experiments, on somatodendritic dopamine release with in vivo microdialysis and on the activation of dopaminergic cells with patch-clamp electrophysiology, provided evidence for a predominant role of GHS-R1a:GHS-R1b:D1R oligomers in the rodent VTA as main mediators of the dopaminergic effects of ghrelin.SIGNIFICANCE STATEMENT The activation of ghrelin receptors localized in the ventral tegmental area (VTA) plays a significant role in the contribution of ghrelin to motivated behavior. We present evidence that indicates these receptors form part of oligomeric complexes that include the functional ghrelin receptor GHS-R1a, its truncated nonsignaling isoform GHS-R1b, and the dopamine D1 receptor (D1R). The binding of ghrelin to these complexes promotes activation of the dopaminergic neurons of the VTA by activation of adenylyl cyclase–protein kinase A signaling, which can be counteracted by both GHS-R1a and D1R antagonists. Our study provides evidence for a predominant role of GHS-R1a:GHS-R1b:D1R oligomers in rodent VTA as main mediators of the dopaminergic effects of ghrelin.

Molecular recognition of an acyl-peptide hormone and activation of ghrelin receptor

Ghrelin, also called “the hunger hormone”, is a gastric peptide hormone that regulates food intake, body weight, as well as taste sensation, reward, cognition, learning and memory. One unique feature of ghrelin is its acylation, primarily with an octanoic acid, which is essential for its binding and activation of the ghrelin receptor, a G protein-coupled receptor. The multifaceted roles of ghrelin make ghrelin receptor a highly attractive drug target for growth retardation, obesity, and metabolic disorders. Here we present two cryo-electron microscopy structures of Gq-coupled ghrelin receptor bound to ghrelin and a synthetic agonist, GHRP-6. Analysis of these two structures reveals a unique binding pocket for the octanoyl group, which guides the correct positioning of the peptide to initiate the receptor activation. Together with mutational and functional data, our structures define the rules for recognition of the acylated peptide hormone and activation of ghrelin receptor, and provide structural templates to facilitate drug design targeting ghrelin receptor.

Characterization of cachexia in the human fibrosarcoma HT-1080 mouse tumour model.

BackgroundCancer cachexia is a complex metabolic disease with unmet medical need. Although many rodent models are available, none are identical to the human disease. Therefore, the development of new preclinical models that simulate some of the physiological, biochemical, and clinical characteristics of the human disease is valuable. The HT‐1080 human fibrosarcoma tumour cell line was reported to induce cachexia in mice. Therefore, the purpose of this work was to determine how well the HT‐1080 tumour model could recapitulate human cachexia and to examine its technical performance. Furthermore, the efficacy of ghrelin receptor activation via anamorelin treatment was evaluated, because it is one of few clinically validated mechanisms.MethodsFemale severe combined immunodeficient mice were implanted subcutaneously or heterotopically (renal capsule) with HT‐1080 tumour cells. The cachectic phenotype was evaluated during tumour development, including body weight, body composition, food intake, muscle function (force and fatigue), grip strength, and physical activity measurements. Heterotopic and subcutaneous tumour histology was also compared. Energy balance was evaluated at standard and thermoneutral housing temperatures in the subcutaneous model. The effect of anamorelin (ghrelin analogue) treatment was also examined.ResultsThe HT‐1080 tumour model had excellent technical performance and was reproducible across multiple experimental conditions. Heterotopic and subcutaneous tumour cell implantation resulted in similar cachexia phenotypes independent of housing temperature. Tumour weight and histology was comparable between both routes of administration with minimal inflammation. Subcutaneous HT‐1080 tumour‐bearing mice presented with weight loss (decreased fat mass and skeletal muscle mass/fibre cross‐sectional area), reduced food intake, impaired muscle function (reduced force and grip strength), and decreased spontaneous activity and voluntary wheel running. Key circulating inflammatory biomarkers were produced by the tumour, including growth differentiation factor 15, Activin A, interleukin 6, and TNF alpha. Anamorelin prevented but did not reverse anorexia and weight loss in the subcutaneous model.ConclusionsThe subcutaneous HT‐1080 tumour model displays many of the perturbations of energy balance and physical performance described in human cachexia, consistent with the production of key inflammatory factors. Anamorelin was most effective when administered early in disease progression. The HT‐1080 tumour model is valuable for studying potential therapeutic targets for the treatment of cachexia.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of macimorelin in healthy adults: Results of a single-dose, randomized controlled study

ObjectiveMacimorelin is an orally active ghrelin receptor agonist indicated for the diagnosis of adult growth hormone (GH) deficiency in the United States. This phase 1 study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending doses of macimorelin (including a supratherapeutic dose to be used in a thorough QT trial) in healthy adults. DesignParticipants were randomized to receive macimorelin 0.5, 1.0, or 2.0 mg/kg or placebo in 1 of 3 sequential ascending-dose cohorts. Blood samples for pharmacokinetic and pharmacodynamic assays were collected pre-dose and at specified time points over a 24-h period. Pharmacokinetic parameters assessed included area under the concentration-time curve (AUC), maximum concentration (Cmax) of macimorelin in plasma, time to Cmax (tmax), and terminal elimination half-life (t1/2). Pharmacodynamic assessments evaluated levels of GH, adrenocorticotropic hormone, thyroid-stimulating hormone, cortisol, and prolactin. Safety was assessed based on treatment-emergent adverse events (TEAEs), vital signs, 12‑lead electrocardiograms, and laboratory parameters. ResultsA total of 28 healthy adults were enrolled and completed the study. Macimorelin AUC and Cmax showed less than dose-proportional increases following administration of 0.5 and 1.0 mg/kg. Mean t1/2 was 3.51 h for macimorelin 0.5 and 1.0 mg/kg and 8.29 h for macimorelin 2.0 mg/kg; median tmax occurred at 0.5 to 0.75 h. GH levels increased after dosing, with a tmax of 0.75 h to 1.0 h. Mean GH Cmax was similar with the macimorelin 0.5- and 1.0-mg/kg doses (31.9 and 37.8 ng/mL, respectively) and was ~50% lower with macimorelin 2.0 mg/kg (18.4 ng/mL). Transient increases were observed in adrenocorticotropic hormone, cortisol, and prolactin, which were not dose related. A total of 19 TEAEs were reported in 35.7% (10/28) of participants; all TEAEs were mild or moderate and resolved. A total of 12 drug-related TEAEs were reported in 8 participants. Headache was the most common drug-related TEAE. All doses of macimorelin prolonged mean QTcF by 10 to 11 ms. There were no clinically meaningful changes in vital signs or laboratory parameters. ConclusionsSingle-dose administration of macimorelin 0.5 to 2.0 mg/kg was well tolerated. Macimorelin exposure was less than dose-proportional over the dose range studied. Administration of macimorelin stimulated GH production, with the greatest increases observed in the macimorelin 0.5- and 1.0-mg/kg groups.

Effects of ghrelin receptor activation on forebrain dopamine release, conditioned fear and fear extinction in C57BL/6J mice.

The ghrelin system was previously proposed to mediate an independent branch of the stress response that curbs fear processing. Interestingly, the ghrelin system was also shown to control the activity of midbrain dopamine neurons. Given that dopamine neurons of the ventral tegmental area appear to have a critical role in fear processing, we aimed to investigate their contribution to the effects of ghrelin on fear processing. Our data show that systemic administration of the ghrelin receptor agonist MK0677, in a dose that induces food intake, has no significant effect on auditory fear processing and does not significantly affect dopamine release in the nucleus accumbens of male C57BL/6J mice. Local administration of the ghrelin receptor agonist MK0677 into the ventral tegmental area significantly increases food intake and it also significantly increased dopamine release in the nucleus accumbens, the medial prefrontal cortex and the amygdala. Nevertheless, it did not significantly affect auditory fear extinction. Our data indicate that pharmacological activation of midbrain dopamine neurons using a ghrelin receptor agonist does not affect auditory fear extinction. We also investigated the effect of non-pharmacological manipulation of the ghrelin system on auditory fear processing. However, we found that neither overnight food deprivation nor genetic ablation of the ghrelin receptor had a significant effect on auditory fear extinction. We conclude that the effects of manipulation of the ghrelin system on fear processing are subject to boundary conditions that remain poorly understood.

The brain‐penetrating, orally bioavailable, ghrelin receptor agonist HM01 ameliorates motion‐induced emesis in Suncus murinus (house musk shrew)

HM01, a novel, orally bioavailable, brain-penetrating agonist of ghrelin receptors, ameliorates emesis in Suncus murinus. This study compared HM01's activity against motion sickness with that of the less brain-penetrating ghrelin receptor agonist, HM02. The potential of HM01 and HM02 to relax isolated mesenteric arteries and to increase feeding was investigated. Radio telemetry was used to record gastric slow waves and body temperature. Plethysmography was used to measure respiratory function. HM01 and HM02 were administered p.o. 1 hr prior to provocative motion, and c-Fos expression in brain sections was assessed. HM01 and HM02 both relaxed precontracted arteries, yielding EC50 values of 2.5 ± 0.5 and 3.5 ± 0.4 nM respectively. HM01 increased feeding, but HM02 did not. Both compounds caused hypothermia and bradygastria. Motion induced 123 ± 24 emetic events. HM01, but not HM02, reduced motion-induced emesis by 67.6%. Motion increased c-Fos expression in the nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMNV), medial vestibular nucleus (MVe), central nucleus of the amygdala, and paraventricular hypothalamic nucleus (PVH). HM01 alone increased c-Fos expression in the area postrema, NTS, DMNV, PVH, and arcuate hypothalamic nucleus; HM02 had a similar pattern except it did not increase c-Fos in the PVH. Both compounds antagonized the motion-induced increases in c-Fos expression in the MVe. HM01 is more effective than HM02 in preventing motion-induced emesis. The difference in potency may relate to activation of ghrelin receptors in the PVH.

OR32-4 Diagnostic Performance of Macimorelin vs the Insulin Tolerance Test (ITT) in 140 Patients at Varying Growth Hormone (GH) Cutpoints: A Post Hoc Analysis

Background: Macimorelin (MAC), an orally active ghrelin receptor agonist, is indicated for the diagnosis of adult growth hormone deficiency (AGHD) in the United States. The efficacy of MAC for AGHD diagnosis was previously demonstrated; comparing MAC at cutpoint values of 2.8 and 5.1 ng/mL with ITT at cutpoint values of 3.0 and 5.1 ng/mL.1,2Objective: This post hoc analysis evaluated the percent agreement and sensitivity and specificity of MAC vs ITT over a range of GH cutpoints. Methods: This analysis included data from a phase 3, open-label, randomized, 2-way crossover study of MAC vs ITT in subjects with high (Group A, n=38), intermediate (Group B, n=37), and low (Group C, n=40) likelihood for AGHD and healthy, matched controls (Group D, n=25).1 Percent agreement (negative, positive, and overall) and estimated sensitivity and specificity were determined using GH cutpoint values of 2.8, 4, 5.1, and 6.5 ng/mL for both MAC and ITT. Results: 74 subjects were classified as GH deficient, and 66 subjects were classified as GH sufficient. These subjects were classified based on the ITT using a cutpoint of 5.1 ng/mL.1 The highest negative, positive, and overall agreements between tests were observed when GH cutpoints chosen for MAC and ITT were identical to each other and were either 2.8 or 5.1 ng/mL. With a GH cutpoint value of 2.8 ng/mL for both tests, negative agreement was 94% (95% CI: 86%, 98%), positive agreement was 87% (95% CI: 76%, 94%), and overall agreement was 91% (95% CI: 85%, 95%). At a GH cutpoint value of 5.1 ng/mL for both tests, negative agreement was 92% (95% CI: 83%, 97%), positive agreement was 82% (95% CI: 72%, 90%), and overall agreement was 87% (95% CI: 80%, 92%). Assuming all Group A participants were cases and all Group D participants were controls, estimated specificities of MAC and ITT were identical (96%) at GH cutpoint values of 2.8, 4, or 5.1 ng/mL. Estimated sensitivity for ITT at GH cutpoint value of 5.1 ng/mL (97%) was higher than for MAC at cutpoint value of 2.8 ng/mL (87%); increasing the test cutpoint to 6.5 ng/mL increased sensitivity to 97% and 100% for MAC and ITT, respectively, but at the expense of specificity decreases to 92% (MAC) and 88% (ITT). Conclusions: Among the cutpoints examined, agreement between MAC and ITT was highest at either 2.8 or 5.1 ng/mL, with positive agreement declining modestly at the higher cutpoint. Sensitivity of MAC was maximal at 6.5 ng/mL but at the expense of a decline in specificity from 96% to 92%, which may be undesirable if the primary consideration is minimization of false-positive diagnosis of AGHD. A MAC cutpoint of 5.1 ng/mL provides maximal specificity (96%) and high sensitivity (92%) with good overall agreement to ITT at the same cutpoint (87%), making it clinically useful for the diagnosis of AGHD. Reference: 1. Garcia JM, et al. J Clin Endocrinol Metab. 2018;103(8):3083-3093. 2. Garcia JM, et al. Presented at ENEA; 17-20 October 2018; Wroclaw, Poland.

SAT-428 Effect of Age, Body Mass Index, and Sex on the Performance of Macimorelin for the Diagnosis of Adult Growth Hormone Deficiency: A Post Hoc Analysis

Background: The diagnostic performance of macimorelin (MAC), an orally active ghrelin receptor agonist approved for the diagnosis of adult growth hormone deficiency (AGHD) in the United States, has previously been demonstrated in a phase 3 study that compared MAC with the insulin tolerance test (ITT).1 However, it is not known if the diagnostic performance of MAC varies according to patient demographics that are known to influence peak growth hormone release induced by other secretagogues. Objective: To determine whether the performance of the MAC diagnostic test is affected by age, baseline body mass index (BMI), and sex. Methods: This post hoc analysis included data from a previously published phase 3 study of subjects with a high likelihood of GHD (Group A, considered to have AGHD based on having a low IGF-I level plus either of these: at least 3 other pituitary hormone deficiencies or a structural hypothalamic or pituitary lesion) vs healthy controls (Group D).1 The probability of AGHD was estimated using 4 logistic models fitted to the data: unadjusted, age-adjusted, baseline BMI-adjusted, and sex-adjusted. Each model considers all subjects as independent observations, not taking matching into account. The area under the curve (AUC) of the estimated receiver operating characteristic (ROC) curve (0 to 1 range, where 1 is perfect) from each adjusted model was compared with the AUC from the unadjusted model. The estimated sensitivity and specificity for each model at cutpoint values of 2.8 and 5.1 ng/mL were calculated. Results: Of the 70 subjects included in the analysis, 41 had a high likelihood of GHD and 29 were healthy controls; mean±SD (range) age was 41.7±13.9 (18-66) years, mean±SD (range) BMI was 27.1±4.0 (20.8-36.6) kg/m2, and 56% were male. The ROC AUC for the unadjusted model was 0.9924 (95% CI: 0.9807, 1), age-adjusted ROC AUC was 0.9924 (95% CI: 0.9807, 1), BMI-adjusted ROC AUC was 0.9916 (95% CI: 0.9786, 1), and sex-adjusted ROC AUC was 0.9950 (95% CI: 0.9861, 1). For the unadjusted model at cutpoint values of 2.8 and 5.1, estimated sensitivity was 88% and 93%, and specificity was 97% and 97%. These values remained the same for cutpoint 2.8 when adjusting for age and adjusting for mean or median BMI. At cutpoint 5.1, the values remained the same when adjusting for age and mean BMI. When adjusting for sex, sensitivity for females was 88% and specificity was 93% at cutpoint 2.8, and sensitivity for females was 94% and specificity was 93% at cutpoint 5.1. Sensitivity for males was 88% and specificity was 100% at cutpoint 2.8, and sensitivity for males was 92% and specificity was 100% at cutpoint 5.1. Conclusions: Results of this post hoc analysis show that the diagnostic performance of MAC was not meaningfully affected by age, baseline BMI, or sex over the ranges that were studied. Reference: 1. Garcia JM, et al. J Clin Endocrinol Metab. 2018;103(8):3083-3093.

119 Staying Above Water: The Impact of Testosterone and Growth Hormone Secretagogues on Fluid Retention Parameters

Although fluid retention is a known side effect of both testosterone therapy (TTh) and growth hormone secretagogue supplementation (GHS), it has never been quantified or studied objectively. The mechanisms of action underlying TTh have been well described while GHS represent a relatively new class of medications that manipulate growth hormone secretion directly and via ghrelin receptor activity. This study utilizes impedence-based body composition analysis to determine the effect of TTh and GHS on fluid retention parameters (FRPs) in a cohort of hypogonadal men. To quantify the amount of water retention caused by TTh and GHS using body composition analysis. Men presenting to a single andrology clinic from 2015 to 2017 were evaluated while on therapy using InBody (IB) impedance-based body composition analysis at baseline and at follow-up visits at least three months apart. The study cohort was comprised of hypogonadal men receiving TTh and GHS or TTh alone. Multivariate analysis was used to determine changes in fluid retention parameters over time controlling for baseline body composition, medication type and duration of use. Fluid retention parameters included total body water (TBW), intracellular water (ICW), and extracellular water (ECW).

A stable meta-carborane enables the generation of boron-rich peptide agonists targeting the ghrelin receptor.

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron-containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein-coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor-directed boron uptake. In this study, we present the generation of short, boron-rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super-agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta-carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron-loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.

Intrarenal ghrelin receptor inhibition ameliorates angiotensin II-dependent hypertension in rats.

The intrarenal ghrelin receptor (GR) is localized to collecting duct (CD) cells, where it increases epithelial Na+ channel (αENaC)-dependent sodium reabsorption in rodents. We hypothesized that chronic GR inhibition with intrarenal GR siRNA lowers blood pressure (BP) in angiotensin II-dependent hypertension via reductions in αENaC-dependent sodium reabsorption. Uninephrectomized Sprague-Dawley rats ( n = 121) received subcutaneous osmotic pumps for chronic systemic delivery of angiotensin II or vehicle (5% dextrose in water). Rats also received intrarenal infusion of vehicle, GR siRNA, or scrambled (SCR) siRNA. In rats receiving intrarenal vehicle or intrarenal SCR siRNA, systemic angiotensin II infusion increased sodium retention and BP on day 1, and BP remained elevated throughout the 5-day study. These rats also demonstrated increased CD GR expression after 5 days of infusion. However, intrarenal GR siRNA infusion prevented angiotensin II-mediated sodium retention on day 1, induced a continuously negative cumulative sodium balance compared with angiotensin II alone, and reduced BP chronically. Glomerular filtration rate and renal blood flow remained unchanged in GR siRNA-infused rats. Systemic angiotensin II infusion also increased serum aldosterone levels, CD αENaC, and phosphorylated serum and glucocorticoid-inducible kinase 1 expression in rats with intrarenal SCR siRNA; however, these effects were not observed in the presence of intrarenal GR siRNA, despite exposure to the same systemic angiotensin II. These data demonstrate that chronic inhibition of intrarenal GR activity significantly reduces αENaC-dependent sodium retention, resulting in a negative cumulative sodium balance, thereby ameliorating angiotensin II-induced hypertension in rats. Renal GRs represent a novel therapeutic target for the treatment of hypertension and other sodium-retaining states.

Abstract P436: Intrarenal Ghrelin Receptor Inhibition Prevents Sodium Retention and Reduces Blood Pressure in Angiotensin II-induced Hypertension

The renal ghrelin receptor (GR) is localized to collecting duct (CD) cells where it increases E Na C-dependent sodium (Na + ) reabsorption. We have previously shown that in uninephrectomized Sprague-Dawley (SD) rats, systemic Ang II infusion induces hypertension after 1 day (129.2 ± 5 vs. 158.2 ± 2.6 mmHg; P<0.01), and this increase is sustained over a 6 day period (191.2 ± 4.0 mmHg; P<0.001). The increase in BP is attenuated with concomitant intrarenal (IR) GR siRNA infusion (F=28.9; P<0.001), but the mechanism is unknown. In this study, we tested whether the reduction in Ang II-induced hypertension in the presence of IR GR siRNA is the result of the prevention of antinatriuresis. Uninephrectomized SD rats (N=33) received a subcutaneous osmotic pump for chronic systemic delivery of Ang II (200 ng/kg/min) or 5% dextrose in water (D 5 W). Rats received IR bolus infusion of D 5 W, GR siRNA (15 μg x 3 over 6 days), or scrambled siRNA (SCR siRNA, 15 μg x 3 over 6 days). Systemic Ang II + SCR siRNA reduced 24 h U Na V from 0.90 ± 0.04 to 0.34 ± 0.08 μmol/min (P<0.001) and increased cumulative Na + balance from 0 to 0.54 mEq Na + (P<0.01) on day 1, with no significant differences for either group for the remainder of the study. IR GR siRNA infusion prevented Ang II-mediated Na + retention (0.86 ± 0.06 vs. 0.87 ± 0.06 μmol/min) and induced a decrease in cumulative Na + balance from 0 to -0.37 ± 0.08 mEq on day 1 with no significant differences for either group for the remainder of the study. At 24 h of Ang II + SCR siRNA infusion, IR GR expression was increased in whole cell kidney (0.55 ± 0.03 vs. 0.91 ± 0.08 DU; P<0.05) and major CD Na + transporter E Na C translocated from intracellular sites to apical plasma membranes to mediate Na + reabsorption (0.34 ± 0.05 vs. 0.51 ± 0.02 DU; P<0.05). In rats receiving IR GR siRNA infusion, however, Ang II failed to recruit E Na C to the apical plasma membranes of CD cells. There was no change in total whole cell E Na C for all conditions. These data demonstrate that chronic inhibition of IR GR activity significantly reduces E Na C-dependent Na + retention, resulting in a negative cumulative Na + balance, thereby preventing Ang II-induced hypertension in rats. Thus, renal GRs represent a novel therapeutic target for the treatment of hypertension and other Na + -retaining states.

Evidence that central pathways that mediate defecation utilize ghrelin receptors but do not require endogenous ghrelin.

In laboratory animals and in human, centrally penetrant ghrelin receptor agonists, given systemically or orally, cause defecation. Animal studies show that the effect is due to activation of ghrelin receptors in the spinal lumbosacral defecation centers. However, it is not known whether there is a physiological role of ghrelin or the ghrelin receptor in the control of defecation. Using immunohistochemistry and immunoassay, we detected and measured ghrelin in the stomach, but were unable to detect ghrelin by either method in the lumbosacral spinal cord, or other regions of the CNS. In rats in which the thoracic spinal cord was transected 5 weeks before, the effects of a ghrelin agonist on colorectal propulsion were significantly enhanced, but defecation caused by water avoidance stress (WAS) was reduced. In knockout rats that expressed no ghrelin and in wild‐type rats, WAS‐induced defecation was reduced by a ghrelin receptor antagonist, to similar extents. We conclude that the ghrelin receptors of the lumbosacral defecation centers have a physiological role in the control of defecation, but that their role is not dependent on ghrelin. This implies that a transmitter other than ghrelin engages the ghrelin receptor or a ghrelin receptor complex.

Mole ghrelin: cDNA cloning, gene expression, and diverse molecular forms in Mogera imaizumii

Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1–27], [1–19], [1–16] and [1–15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5′-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals.

Identification of Novel Probiotics to Modify Appetite and Satiety Directly Targeting the Ghrelin Receptor

Ghrelin is a stomach‐derived peptide that acts as orexigenic hormone in the hypothalamus stimulating food intake through the growth hormone secretagogue receptor (GHS‐R1a). Dysregulated ghrelin signalling may contribute to the development of metabolic disorders such as obesity. Current pharmacologic anti‐obesity treatments lack efficacy and have shown severe side effects, highlighting the urgent need for novel strategies contributing to the maintenance of a healthy weight.Natural products are receiving special consideration as sources of bioactives with beneficial health effects which may be safer for consumers than synthetic therapeutics. One of the most investigated natural bioactives are live microorganisms with health benefit effects, also known as probiotics. Thus, food products containing probiotics are receiving increased scientific and commercial focus in recent years. These probiotic‐mediated beneficial effects on human health are due to changes in the gut microbiota via oral delivery of viable strains of bacteria that are then integrated into the gut system. Gut microbiota ferment nutrients and secrete bioactive compounds inducing several responses within the intestinal mucosa and controlling the gut's barrier and endocrine functions.In this study we investigate the ability of different probiotic bacteria to modulate the GHS‐R1a receptor. Bacteria were cultured overnight under anaerobic conditions at 37°C. Culture supernatants were collected by centrifugation, resuspended in 1× Hanks balanced salt solution containing 20mM HEPES, and incubated in anaerobiosis for 4 h. Afterwards, supernatants were used to investigate the probiotics ghrelin receptor activation potential by calcium mobilization and receptor internalization assays in human embryonic kidney cells (Hek) stably expressing the GHS‐R1a receptor.Different Lactobacillus and Bifidobacterium strains with ability to modulate the ghrelin receptor have been identified. Future investigations, including identification of the bioactives present in the probiotic supernatants as well as in vivo studies supporting these findings, are needed in order to confirm their potential as anti‐obesity therapeutics.Support or Funding InformationWork funded by CREMO SA

Ghrelin receptor (GHS-R1A) antagonism alters preference for ethanol and sucrose in a concentration-dependent manner in prairie voles

Ghrelin receptor (GHS-R1A) activity has been implicated in reward for preferred foods and drugs; however, a recent study in our laboratory indicated that GHS-R1A antagonism reduces early (after only four exposures) preference for 20% ethanol, but not 10% sucrose in prairie voles, a genetically diverse high alcohol-consuming species. The purpose of the present study was to determine if these effects of GHS-R1A antagonism depend on the concentration of the rewarding solution being consumed. We first characterized preference for varying concentrations of ethanol and sucrose. Two bottle tests of each ethanol concentration versus water indicated that 10% and 20% ethanol are less preferred than 3% ethanol, and a follow-up direct comparison of 10% vs. 20% showed that 10% was preferred over 20%. Direct two-bottle comparisons of 2% vs. 5%, 2% vs. 10%, and 5% vs. 10% sucrose showed that 10% sucrose was most preferred, and 2% sucrose was least preferred. The effects of JMV 2959, a GHS-R1A antagonist, on preference for each concentration of ethanol and sucrose were then tested. In a between groups design prairie voles were given four two-hour drinking sessions in which animals had access to ethanol (3, 10, or 20%) versus water, or sucrose (2, 5, or 10%) versus water every other day. Saline habituation injections were given 30min before the third drinking session. JMV 2959 (i.p.; 9mg/kg), a GHS-R1A antagonist, or saline was administered 30min before the fourth drinking session. JMV 2959 reduced preference for 20% ethanol and 2% sucrose, but had no significant effect on preference for the other ethanol and sucrose concentrations. These data identify constraints on the role of GHS-R1A in early preference for ethanol and sucrose, and the concentration-dependent effects suggest strong preference for a reward may limit the importance of GHS-R1A activity.

Blunted hypothalamic ghrelin signaling reduces diet intake in rats fed a low-protein diet in late pregnancy.

Diet intake in pregnant rats fed a low-protein (LP) diet was significantly reduced during late pregnancy despite elevated plasma levels of ghrelin. In this study, we hypothesized that ghrelin signaling in the hypothalamus is blunted under a low-protein diet condition and therefore, it does not stimulate diet intake during late pregnancy. Female Sprague-Dawley rats were fed a normal (CT) or LP diet from Day 1 of pregnancy. On Day 21, 0.5μg ghrelin was given into the third ventricle (ICV). Diet and water intake at 30, 60, and 120min after ICV injection was measured. Hypothalami were dissected and analyzed for expression of genes related to appetite regulation (Npy, Agrp, Pomc and Cart) and phosphorylation of AMPK and ACC proteins (downstream proteins of ghrelin receptor activation). Results include: In response to ICV injection of ghrelin, (1) diet intake was significantly lower in LP compared to CT rats; (2) water intake was not affected in LP rats; (3) expression of Npy and Agrp, but not Pomc and Cart, were higher in the hypothalamus of LP compared to CT rats; (4) the abundance of phosphorylated AMPK and the ratio of phosphorylated to total AMPK, but not the abundance of total AMPK, were lower in LP compared to CT rats; (5) the abundance of phosphorylated ACC, but not total ACC, was lower in LP rats. These findings suggest that blunted ghrelin signaling in the hypothalamus of pregnant rats fed a LP diet leads to reduced diet intake and exacerbates gestational protein insufficiency.

Ghrelin receptor activity amplifies hippocampal N-methyl-d-aspartate receptor-mediated postsynaptic currents and increases phosphorylation of the GluN1 subunit at Ser896 and Ser897.

Although ghrelin and its cognate receptor growth hormone secretagogue receptor (GHSR1a) are highly localized in the hypothalamic nuclei for the regulation of metabolic states and feeding, GHSR1a is also highly localized in the hippocampus, suggesting its involvement in extra-hypothalamic functions. Indeed, exogenous application of ghrelin has been reported to improve hippocampal learning and memory. However, the underlying mechanism of ghrelin regulation of hippocampal functions is poorly understood. Here, we report ghrelin-promoted phosphorylation of GluN1 and amplified N-methyl-d-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents in the CA1 pyramidal cells of the hippocampus in slice preparations. The ghrelin-induced responses were sensitive to a GHSR1a antagonist and inverse agonist, and were absent in GHSR1a homozygous knock-out mice. These results indicated that activation of GHSR1a was critical in the ghrelin-induced enhancement of the NMDAR function. Interestingly, heterozygous mouse hippocampi were also insensitive to ghrelin treatment, suggesting that a slight reduction in the availability of GHSR1a may be sufficient to negate the effect of ghrelin on GluN1 phosphorylation and NMDAR channel activities. In addition, NMDAR-mediated spike currents, which are of dendritic origin, were blocked by the GHSR1a antagonist, suggesting the presence of GHSR1a on the pyramidal cell dendrites in physical proximity to NMDAR. Together with our findings on the localization of GHSR1a in the CA1 region of the hippocampus, which was shown by fluorescent ghrelin binding, immunoreactivity, and enhanced green fluorescent protein reporter gene expression, we conclude that the activation of GHSR1a favours rapid modulation of the NMDAR-mediated glutamatergic synaptic transmission by phosphorylating GluN1 in the hippocampus.

Cancer Cachexia: One Step Ahead

Cachexia is one of the most common manifestations in advanced cancer patients, but too often it remains under- recognized and under-treated. Starvation is not the same of cachexia. Cachexia is defined by "weight loss >5% over past 6 months in absence of simple starvation or the combination of ongoing weight loss>2% with BMI <20 or sarcopenia". The pathogenesis of cancer cachexia is not fully understood, but inflammation and an increased catabolic response to a number of cancer-related factors seem to represent the basis of any assumption. Early diagnosis of a pre-cachectic or cachectic state is a key moment for the treatment of this complex syndrome, in order to guarantee an adequate food intake and suitable exercise and to interfere with the inflammatory processes that are typical of cachexia. Therefore, one of the main aims is to identify those patients most likely to develop the syndrome early. A multimodality baseline approach to cancer cachexia addresses reversible clinical contributory factors. There are currently no medicinal products that have a proven efficacy in the medical approach to cancer cachexia. Recently, anamorelin, a synthetic orally active ghrelin receptor agonist, showed promising results, but the best approach to cancer cachexia probably remains an early multimodal interventions consisting in nutritional intervention, exercise and rehabilitation program, and multi-target drug therapies. This review summarizes what we know and what still need to know about cancer cachexia syndrome.