Growth Hormone Secretagogue Research Articles

H. pylori effects on ghrelin axis: Preliminary change in gastric pathogenesis

Ghrelin and its receptors are present in the stomach, suggesting that the ghrelin axis plays an essential role in gastrointestinal complications. This investigation aimed to explore the effects of H. pylori infection and gastritis on serum ghrelin and ghrelin axis gene expression. In this study, we enrolled 68 adult ambulatory people referred for upper gastrointestinal endoscopy. The individuals were classified into three groups based on H. pylori infection and gastritis. Total serum ghrelin and tissue gene expression were tested with ELISA and quantitative RT-PCR, respectively. Serum ghrelin and mRNA expression were significantly lower in H. pylori-positive with gastritis subjects compared with both H. pylori-negative with and without gastritis. Growth hormone secretagogue receptor1a mRNA expression was not different between groups while GHSR1b expression was significantly higher in patients with H. pylori infection and gastritis. We propose the ghrelin axis intermediaries, such as GHSR1b, as a potential clinical target for gastric disorders.

The emerging role of heterodimerisation and interacting proteins in ghrelin receptor function.

Ghrelin is a peptide hormone secreted primarily by the stomach that acts upon the growth hormone secretagogue receptor (GHSR1), a G protein-coupled receptor whose functions include growth hormone secretion, appetite regulation, energy expenditure, regulation of adiposity, and insulin release. Following the discovery that GHSR1a stimulates food intake, receptor antagonists were developed as potential therapies to regulate appetite. However, despite reductions in signalling, the desired effects on appetite were absent. Studies in the past 15 years have demonstrated GHSR1a can interact with other transmembrane proteins, either by direct binding (i.e. heteromerisation) or via signalling cross-talk. These interactions have various effects on GHSR1a signalling including preferential coupling to one pathway (i.e. biased signalling), coupling to a unique G protein (G protein switching), suppression of GHSR1a signalling, and enhancement of signalling by both receptors. While many of these interactions have been shown in cells overexpressing the proteins of interest and remain to be verified in tissues, substantial evidence exists showing that GHSR1a and the dopamine receptor D1 (DRD1) form heteromers, which promote synaptic plasticity and formation of hippocampal memory. Additionally, a reduction in GHSR1a-DRD1 complexes in favour of establishment of GHSR1a-Aβ complexes correlates with Alzheimer’s disease, indicating that GHSR1a heteromers may have pathological functions. Herein, we summarise the evidence published to date describing interactions between GHSR1a and transmembrane proteins, discuss the experimental strengths and limitations of these studies, describe the physiological evidence for each interaction, and address their potential as novel drug targets for appetite regulation, Alzheimer’s disease, insulin secretion, and inflammation.

Characterization of equine liver microsome-generated metabolites of growth hormone secretagogue small molecule ibutamoren.

Interest in growth hormone secretagogues has intensified during the past several years based on capable, ever-widening investigational applications of recombinant growth hormone in animals and humans. Ibutamoren is a potent, long-acting, selective and orally active non-peptide growth hormone secretagogue, which has a great potential for abuse as a performance-enhancing agent in sports. To support drug metabolism and pharmacokinetic studies of chiral pharmaceuticals, it is necessary to combine the resolving power of high-performance liquid chromatography with the sensitivity of mass spectrometric techniques. This paper describes the metabolic conversion of ibutamoren using equine liver microsomes and metabolite characterization using a QExactive high-resolution mass spectrometer. A total of 32 metabolites for ibutamoren (20 phase I and 12 phase II) were detected. The important findings of the current research are as follows: (1) the growth hormone secretagogue ibutamoren was prone to oxidation, resulting in corresponding hydroxylated metabolites; (2) in ibutamoren, the dissociation of the phenyl ring and 2-amino-2-methylpropanamide side chain was also observed; (3) the glucuronic acid conjugates of mono-, di- and trihydroxylated analogues were detected; and (4) no sulfonic acid conjugated metabolites were observed in this study of ibutamoren. The reported data help in the speedy detection of the growth hormone secretagogue ibutamoren and reveal its illegal use in competitive sports.

Protective and Healing Effects of Ghrelin and Risk of Cancer in the Digestive System.

Ghrelin is an endogenous ligand for the ghrelin receptor, previously known as the growth hormone secretagogue receptor. This hormone is mainly produced by endocrine cells present in the gastric mucosa. The ghrelin-producing cells are also present in other organs of the body, mainly in the digestive system, but in much smaller amount. Ghrelin exhibits a broad spectrum of physiological effects, such as stimulation of growth hormone secretion, gastric secretion, gastrointestinal motility, and food intake, as well as regulation of glucose homeostasis and bone formation, and inhibition of inflammatory processes. This review summarizes the recent findings concerning animal and human data showing protective and therapeutic effects of ghrelin in the gut, and also presents the role of growth hormone and insulin-like growth factor-1 in these effects. In addition, the current data on the possible influence of ghrelin on the carcinogenesis, its importance in predicting the risk of developing gastrointestinal malignances, as well as the potential usefulness of ghrelin in the treatment of cancer, have been presented.

Early detection of cannabinoids in biological samples based on their affinity interaction with the growth hormone secretagogue receptor

Herein we report on the early detection of cannabinoids in urine samples according to their affinity profiles in competitive assays with labelled ghrelin (GHR). We have demonstrated for the first time that cannabidiol (CBD) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC) act as extracellular ligands for the growth hormone secretagogue receptor (GHS-R1a), strongly promoting the binding of ghrelin (GHR), the endogenous ligand of GHS-R1a. The affinity profiles of CBD and carboxy-THC are significantly different from the profiles of synthetic GHR mimetics such as CJC-1295 or [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-Substance P peptides, which are the most common interferents; the cannabinoids promoted the GHR/GHS-R1a interaction, while the ghrelin mimetics acted rather as competitive inhibitors. The analysis of 1:4 diluted urine samples proved that the proposed method displays good linearity and sensitivity in the range of 5–30 ng/mL for both CBD and carboxy-THC, whereas GHR mimetics display no interference at concentrations up to 100 ng/mL. The results were validated by comparison with the gas chromatography tandem mass spectrometry reference method. CBD may exert the same promoting effect on the interaction of GHS-R1a with other GHR mimetics listed as performance-enhancing substances.

Growth hormone secretagogue peptide-6 enhances oreochromicins transcription and antimicrobial activity in tilapia (Oreochromis sp.)

Growth Hormone-Releasing Peptide 6 (GHRP-6) (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2) is an agonist of the growth hormone secretagogue receptor. GHRP-6 mimics the effect of ghrelin. The present study focuses on the immunomodulatory effects of GHRP-6 in tilapia with and without the presence of Pseudomonas aeruginosa infection. GHRP-6 up-regulated the transcription levels of three piscidin-like antimicrobial peptides (Oreochromicins I, II, and III) and granzyme in a tissue-dependent manner. Antimicrobial activity stimulation in serum (lysozyme and anti-protease activity) was also confirmed. Besides, GHRP-6 enhanced the in vitro antimicrobial activity against P. aeruginosa in tilapia gills mucus and serum samples and decreased the bacterial load in vivo after infection with this Gram-negative bacterium. Our results evidenced, for the first time, a direct link between a growth hormone secretagogue ghrelin mimetic in fish and the enhancement of antimicrobial peptides transcription, which suggests that this secretagogue is capable to lead the activation of microbicidal activity in tilapia. Thus, these results open new possibilities for GHRP-6 application in aquaculture to stimulate the teleost immune system as an alternative treatment against opportunistic bacteria.

Changes in energy metabolism and respiration in different\xa0tracheal narrowing in rats

Why obstructive sleep apnea (OSA) treatment does not completely restore healthy metabolic physiology is unclear. In rats, the need for respiratory homeostasis maintenance following airway obstruction (AO) is associated with a loss of thermoregulation and abnormal metabolic physiology that persists following successful obstruction removal. Here, we explored the effect of two different types of tracheal narrowing, i.e., AO and mild airway obstruction (mAO), and its removal on respiratory homeostasis and metabolic physiology. We show that after ten weeks, mAO vs. AO consumes sufficient energy that is required to maintain respiratory homeostasis and thermoregulation. Obstruction removal was associated with largely irreversible increased feeding associated with elevated serum ghrelin, hypothalamic growth hormone secretagogue receptor 1a, and a phosphorylated Akt/Akt ratio, despite normalization of breathing and energy requirements. Our study supports the need for lifestyle eating behavior management, in addition to endocrine support, in order to attain healthy metabolic physiology in OSA patients.

Yeokwisan, a Standardized Herbal Formula, Enhances Gastric Emptying via Modulation of the Ghrelin Pathway in a Loperamide-induced Functional Dyspepsia Mouse Model.

Background: Yeokwisan, a standardized herbal formula, has exhibited clinical benefit for patients suffering from refractory functional dyspepsia (FD) in Korea since 2016. However, data about the mechanism of action of this formula are yet not available.Aim of the study: To evaluate and explore the effects of Yeokwisan on gastric emptying, a major symptom of functional dyspepsia, and its underlying mechanisms of action using a mouse model.Materials and methods: BALB/C mice were pretreated with Yeokwisan (100, 200, and 400 mg/kg, po) or mosapride (3 mg/kg, po) for 5 days and then treated with loperamide (10 mg/kg, ip) after 20 h of fasting. A solution of 0.05% phenol red (500 μL) or diet of 5% charcoal (200 μL) was orally administered, followed by assessment of gastric emptying or intestinal transit. Plasma acyl-ghrelin (ELISA), C-kit (immunofluorescence and western blotting), nNOS (western blotting) and gastric contraction- and ghrelin-related gene/protein expression levels were examined in stomach and small intestine tissues.Results: Loperamide injection substantially delayed gastric emptying, while Yeokwisan pretreatment (especially 200 and 400 mg/kg Yeokwisan) significantly attenuated this peristaltic dysfunction, as evidenced by the quantity of phenol red retained in the stomach (p < 0.05 or 0.01) and stomach weight (p < 0.05 or 0.01). The levels of plasma acyl-ghrelin and expression of gastric ghrelin-related genes, such as growth hormone secretagogue receptor (GHSR), ghrelin-O-acyltransferase (GOAT), adrenergic receptor β1 (ADRB1) and somatostatin receptor (SSTR), were significantly normalized (p < 0.05 or 0.01) by Yeokwisan (400 mg/kg). Yeokwisan (400 mg/kg) significantly tempered the loperamide-induced alterations in the c-kit and nNOS levels (p < 0.01) as well as the expression of contraction- and ghrelin-related genes, such as 5-HT4 receptor (5-HT4R), anoctamin-1 (ANO1), ryanodine receptor 3 (RYR3) and smooth muscle myosin light chain kinase (smMLCK), in the stomach, but not in the small intestine.Conclusion: The present results showed the clinical relevance of Yeokwisan, in treating FD, especially in promoting gastric emptying but not small intestinal transit. The main mechanisms corresponding to these effects may involve the modulation of the ghrelin pathway and activation of interstitial cells of Cajal in stomach tissue.

GHSR methylation-dependent expression of a variant ligand and receptor of the ghrelin system induces thymoma tumorigenesis.

Our previous study reported that the DNA methylation of growth hormone secretagogue receptor (GHSR) was significantly higher in thymoma or thymic carcinoma (TC) than in normal thymic tissue samples. Thymic epithelial tumors (TETs) with higher GHSR DNA methylation were associated with significantly worse prognosis than those with lower levels of DNA methylation. Diversified components of the ghrelin-GHSR axis may exert opposing effects in cancer progression, depending on the cancer type in question. However, the precise function of the axis remains unclear. In the present study, the mRNA expression of five key components of the ghrelin system [native ligand ghrelin, variant ligand In-1 ghrelin, native receptor GHSR1a, variant receptor GHSR1b and acylation enzyme ghrelin O-acyltransferase (GOAT)] were examined in 58 TET samples by reverse transcription-quantitative PCR, and protein expression of GHSR1a and GHSR1b was assessed in 20 TETs using immunohistochemistry. The results revealed that In-1 ghrelin, GHSR1b (variant forms) and GOAT were more strongly expressed in thymoma compared with thymic-adjacent tissue. By contrast, no significant differences were observed in the expression of ghrelin and GHSR1a (native forms) between thymoma and thymic tissue. The mRNA expression of In-1 ghrelin and GHSR1b (variant forms) was positively associated with GHSR methylation in thymoma tissue samples. However, a relationship was not found between ghrelin, GHSR1a or GOAT expression (native forms) and GHSR methylation in thymoma. Immunohistochemical analysis revealed that mRNA expression of GHSR1a and GHSR1b generally correlated with expression of the corresponding protein, and that the expression of GHSR1b was increased in advanced-stage TETs. These results indicate that the DNA methylation of GHSR is associated with a shift from native expression (ghrelin and GHSR1a) to variant expression (In-1 ghrelin and GHSR1b), which induces the tumorigenesis of thymoma, but not TC.

Liver-Expressed Antimicrobial Peptide 2 antagonizes the insulinostatic effect of ghrelin in rat isolated pancreatic islets.

The hormone ghrelin is the endogenous agonist of the G protein-coupled receptor (GPCR) termed growth hormone secretagogue receptor (GHSR). Ghrelin inhibits glucose-stimulated insulin secretion by activating pancreatic GHSR. Recently, Liver-Expressed Antimicrobial Peptide 2 (LEAP2) was recognized as an endogenous GHSR ligand that blocks ghrelin-induced actions. Nonetheless, the effect of LEAP2 on glucose-stimulated insulin secretion from pancreatic islets is unknown. We aimed at exploring the activity of LEAP2 on glucose-stimulated insulin secretion. Islets of Langerhans isolated from rat pancreas were exposed to glucose in the presence or in the absence of LEAP2 and ghrelin and then insulin secretion was assayed. LEAP2 did not modulate glucose-stimulated insulin secretion. However, LEAP2 blocked the insulinostatic action of ghrelin. Our data show that LEAP2 behaves as an antagonist of pancreatic GHSR.

Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor.

There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Dried blood spots, an emerging tool for doping control

The presentation will afford information on the possibilities for dried blood spot (DBS) samples as a new tool supporting the control of the abuse of prohibited substances in the world of sport. Based on previous developments in other biological fields, recent years have seen increasing studies devoted to the possible application of dried drops of blood collected on different support materials as a promising tool to detect the presence of prohibited substances in the body of potential doping athletes. Among the studied analytes, there are examples of most of the groups of substances in the extensive prohibited list of the World Antidoping Agency (WADA). In general terms, the mere presence of most of those prohibited substances in sports drug testing showed by DBS could be enough to be considered as proof of external application to the body, and therefore susceptible to plausible sanctions. However, in other cases, the decision on potential positivity should be based on quantitative criteria (the so-called threshold substances), which is somehow more challenging for DBS. Pre-analytical aspects such exact volume of blood collected in the spots, hematocrit derived effects or homogeneity of the spots may be relevant. Examples of DBS testing for some of the more representative compounds (e.g. testosterone esters, stimulants, erythropoietin, human growth hormone, GH secretagogues, …) analyzed by chromatographic, mass spectrometric, electrophoretic or immunological methods will be presented. Also, considerations related to: – different potential matrices to collect the blood spots, such as cellulose based materials or polymers supports; – collection from finger prick or upper arm; – comparison of capillary vs. venous blood; – use of either manual or device-based collecting approaches; – insight into the degree of acceptability of those different possibilities by athletes, will be commented. Given the strict requirements for procedures in doping control (e.g. WADA and ISO 17025 accreditations), other aspects such as rigorous sample collection supervision (sample A for initial analysis and sample B for counter analysis), transport, analytical procedures and storage are to be aligned with the strictness for other usual samples such as urine or liquid blood in sport drug testing. To this end, a comprehensive Technical Document is being developed by a Working Group and to be approved by the WADA. In a first step, qualitative applications are included. A second step, addressed to threshold substances requiring quantitative estimations, is to be subsequently developed. DBS appear as an emerging new tool for prohibited drugs testing in sport, initially supporting more traditional procedures in the field such as urine or liquid blood analyses, but also susceptible of further developments by itself alone. Advantages in terms of easiness of collection, greater analyte stability and simple transport and storage, probably will overcome limitations such as the reduced amount of available sample and consequent limit in the number of substances detectable from a single DBS. In any case, its emerging applicability in sports drug testing will need strict technical controls.

Circulating ghrelin crosses the blood-cerebrospinal fluid barrier via growth hormone secretagogue receptor dependent and independent mechanisms

Ghrelin is a peptide hormone mainly secreted from gastrointestinal tract that acts via the growth hormone secretagogue receptor (GHSR), which is highly expressed in the brain. Strikingly, the accessibility of ghrelin to the brain seems to be limited and restricted to few brain areas. Previous studies in mice have shown that ghrelin can access the brain via the blood-cerebrospinal fluid (CSF) barrier, an interface constituted by the choroid plexus and the hypothalamic tanycytes. Here, we performed a variety of in vivo and in vitro studies to test the hypothesis that the transport of ghrelin across the blood-CSF barrier occurs in a GHSR-dependent manner. In vivo, we found that the uptake of systemically administered fluorescent ghrelin in the choroid plexus epithelial (CPE) cells and in hypothalamic tanycytes depends on the presence of GHSR. Also, we detected lower levels of CSF ghrelin after a systemic ghrelin injection in GHSR-deficient mice, as compared to WT mice. In vitro, the internalization of fluorescent ghrelin was reduced in explants of choroid plexus from GHSR-deficient mice, and unaffected in primary cultures of hypothalamic tanycytes derived from GHSR-deficient mice. Finally, we found that the GHSR mRNA is detected in a pool of CPE cells, but is nearly undetectable in hypothalamic tanycytes with current approaches. Thus, our results suggest that circulating ghrelin crosses the blood-CSF barrier mainly by a mechanism that involves the GHSR, and also possibly via a GHSR-independent mechanism.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Macimorelin in Children with Suspected Growth Hormone Deficiency: An Open-Label, Group Comparison, Dose-Escalation Trial

Background/Aims: Diagnosis of growth hormone deficiency (GHD) in children requires the use of provocative growth hormone (GH) stimulation tests, which can have limited reliability and are potentially contraindicated in some patients. This is the first paediatric study to test the safety, tolerability, and pharmacokinetics (PK)/pharmacodynamics (PD) of macimorelin, an oral GH secretagogue, approved for diagnosis of adult GHD. Methods: In this open-label, group comparison, single-dose escalation trial (EudraCT 2018-001988-23), sequential cohorts of patients (C1–C3) received ascending single doses of macimorelin: 0.25 (C1), 0.5 (C2), and 1.0 (C3) mg/kg. Primary endpoints were safety and tolerability, and secondary endpoints were PK/PD. Results: Twenty-four patients aged between 2 and <18 with suspected GHD participated in the study. No macimorelin-related adverse events were reported, and macimorelin was well tolerated. Plasma macimorelin concentrations increased with dose: mean areas under the curve were 6.69 (C1), 18.02 (C2), and 30.92 (C3) h × ng/mL; mean maximum concentrations were 3.46 (C1), 8.13 (C2), and 12.87 (C3) ng/mL. GH concentration increased following macimorelin administration: mean times of maximum measured concentration were 52.5 (C1), 37.5 (C2), and 37.5 (C3) min. Conclusion: All 3 doses of macimorelin had excellent safety and tolerability with PK/PD profiles in expected ranges. These results support the use of 1.0 mg/mL macimorelin in a Phase 3 test validation trial in children.

LEAP-2: An Emerging Endogenous Ghrelin Receptor Antagonist in the Pathophysiology of Obesity.

Liver-expressed antimicrobial peptide 2 (LEAP-2), originally described as an antimicrobial peptide, has recently been recognized as an endogenous blocker of growth hormone secretagogue receptor 1a (GHS-R1a). GHS-R1a, also known as ghrelin receptor, is a G protein-coupled receptor (GPCR) widely distributed on the hypothalamus and pituitary gland where it exerts its major functions of regulating appetite and growth hormone (GH) secretion. The activity of GHS-R1a is controlled by two counter-regulatory endogenous ligands: Ghrelin (activation) and LEAP-2 (inhibition). Ghrelin activates GHS-R1a on the neuropeptide Y/Agouti-related protein (NPY/AgRP) neurons at the arcuate nucleus (ARC) to promote appetite, and on the pituitary somatotrophs to stimulate GH release. On the flip side, LEAP-2, acts both as an endogenous competitive antagonist of ghrelin and an inverse agonist of constitutive GHS-R1a activity. Such a biological property of LEAP-2 vigorously blocks ghrelin’s effects on food intake and hormonal secretion. In circulation, LEAP-2 displays an inverse pattern as to ghrelin; it increases with food intake and obesity (positive energy balance), whereas decreases upon fasting and weight loss (negative energy balance). Thus, the LEAP-2/ghrelin molar ratio fluctuates in response to energy status and modulation of this ratio conversely influences energy intake. Inhibiting ghrelin’s activity has shown beneficial effects on obesity in preclinical experiments, which sheds light on LEAP-2’s anti-obesity potential. In this review, we will analyze LEAP-2’s effects from a metabolic point of view with a focus on metabolic hormones (e.g., ghrelin, GH, and insulin), and discuss LEAP-2’s potential as a promising therapeutic target for obesity.

Contribution of Ghrelin to the Pathogenesis of Growth Hormone Deficiency.

In this review we described the interactions between ghrelin and the growth hormone (GH)-insulin-like growth factor 1 (IGF-1) axis in children and adults with growth hormone deficiency (GHD). A possible involvement of these interactions in the pathogenesis of unexplained cases of GHD was suggested. Current research provides more and more details to the knowledge on the circadian rhythm of ghrelin. We gathered reports on the decreasing effect of Helicobacter pylori-related chronic gastritis on the number of ghrelin immunopositive cells and the consequent decrease in ghrelin serum concentration. The gastrointestinal tract microflora modification of the ghrelin action, by the mechanism of molecular mimicry, was also stressed. Moreover, the mutual relationships between ghrelin and the TSH-FT4/FT3 axis in growth and metabolic processes are described. It is to be recalled that FT4 and FT3 exert a permissive impact on IGF-1 action and, in turn, GH, in reaction mediated by IGF-1, enhances the monodeiodination of FT4 to FT3. Finally, we discussed the latest attempts to use the GH secretagogue receptor (GHS-R) analogues for possible diagnostic and therapeutic purposes.

LEAP2 deletion in mice enhances ghrelin's actions as an orexigen and growth hormone secretagogue

ObjectiveThe hormone liver-expressed antimicrobial peptide-2 (LEAP2) is a recently identified antagonist and an inverse agonist of the growth hormone secretagogue receptor (GHSR). GHSR's other well-known endogenous ligand, acyl-ghrelin, increases food intake, body weight, and GH secretion and is lowered in obesity but elevated upon fasting. In contrast, LEAP2 reduces acyl-ghrelin-induced food intake and GH secretion and is found elevated in obesity but lowered upon fasting. Thus, the plasma LEAP2/acyl-ghrelin molar ratio could be a key determinant modulating GHSR signaling in response to changes in body mass and feeding status. In particular, LEAP2 may serve to dampen acyl-ghrelin action in the setting of obesity, which is associated with ghrelin resistance. Here, we sought to determine the metabolic effects of genetic LEAP2 deletion. MethodsWe generated the first known LEAP2-KO mouse line. Food intake, GH secretion, and cellular activation (c-fos induction) in different brain regions following s.c. acyl-ghrelin administration in LEAP2-KO mice and wild-type littermates were determined. LEAP2-KO mice and wild-type littermates were submitted to a battery of tests (such as measurements of body weight, food intake, and body composition; indirect calorimetry, determination of locomotor activity, and meal patterning while housed in metabolic cages) over the course of 16 weeks of high-fat diet and/or standard chow feeding. Fat accumulation was assessed in hematoxylin & eosin-stained and oil red O-stained liver sections from these mice. ResultsLEAP2-KO mice were more sensitive to s.c. ghrelin. In particular, acyl-ghrelin acutely stimulated food intake at a dose of 0.5 mg/kg BW in standard chow-fed LEAP2-KO mice while a 2× higher dose was required by wild-type littermates. Also, acyl-ghrelin stimulated food intake at a dose of 1 mg/kg BW in high-fat diet-fed LEAP2-KO mice while not even a 10× higher dose was effective in wild-type littermates. Acyl-ghrelin induced a 90.9% higher plasma GH level and 77.2–119.7% higher numbers of c-fos-immunoreactive cells in the arcuate nucleus and olfactory bulb, respectively, in LEAP2-KO mice than in wild-type littermates. LEAP2 deletion raised body weight (by 15.0%), food intake (by 18.4%), lean mass (by 6.1%), hepatic fat (by 42.1%), and body length (by 1.7%) in females on long-term high-fat diet as compared to wild-type littermates. After only 4 weeks on the high-fat diet, female LEAP2-KO mice exhibited lower O2 consumption (by 13%), heat production (by 9.5%), and locomotor activity (by 49%) than by wild-type littermates during the first part of the dark period. These genotype-dependent differences were not observed in high-fat diet-exposed males or female and male mice exposed for long term to standard chow diet. ConclusionsLEAP2 deletion sensitizes lean and obese mice to the acute effects of administered acyl-ghrelin on food intake and GH secretion. LEAP2 deletion increases body weight in females chronically fed a high-fat diet as a result of lowered energy expenditure, reduced locomotor activity, and increased food intake. Furthermore, in female mice, LEAP2 deletion increases body length and exaggerates the hepatic fat accumulation normally associated with chronic high-fat diet feeding.

LEAP2 Impairs the Capability of the Growth Hormone Secretagogue Receptor to Regulate the Dopamine 2 Receptor Signaling.

The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (CaV2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on CaV2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of CaV2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on CaV2.2 currents, and that the GHSR-binding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR.

Identification of a Putative β-Arrestin Superagonist of the Growth Hormone Secretagogue Receptor (GHSR).

Ghrelin is a pleiotropic feeding hormone which also has a pivotal role in the central nervous system. Upon the activation of its receptor, growth hormone secretagogue receptor (GHSR), the Gαq/11 -mediated and the β-arrestin-mediated signaling pathways are activated. As the β-arrestin pathway is a potential drug target, there is a strong need for β-arrestin-biased GHSR modulators. Activation of the β-arrestin pathway should inhibit the Gαq/11 -mediated calcium flux through internalization of the receptor. Hence, we used the antagonistic activity in the calcium assay as the first screening for the β-arrestin activation. By conducting the second screening assay for the β-arrestin activation based on extracellular signal regulated kinase (ERK) 1/2 phosphorylation, we discovered a putative β-arrestin-biased superagonist. The activity of the compound was not completely blocked with the competitive antagonist, which implies that the effect is mediated, at least partly, by allosteric binding of the compound.

High Coexpression of the Ghrelin and LEAP2 Receptor GHSR With Pancreatic Polypeptide in Mouse and Human Islets.

Islets represent an important site of direct action of the hormone ghrelin, with expression of the ghrelin receptor (growth hormone secretagogue receptor; GHSR) having been localized variably to alpha cells, beta cells, and/or somatostatin (SST)-secreting delta cells. To our knowledge, GHSR expression by pancreatic polypeptide (PP)-expressing gamma cells has not been specifically investigated. Here, histochemical analyses of Ghsr-IRES-Cre × Cre-dependent ROSA26-yellow fluorescent protein (YFP) reporter mice showed 85% of GHSR-expressing islet cells coexpress PP, 50% coexpress SST, and 47% coexpress PP + SST. Analysis of single-cell transcriptomic data from mouse pancreas revealed 95% of Ghsr-expressing cells coexpress Ppy, 100% coexpress Sst, and 95% coexpress Ppy + Sst. This expression was restricted to gamma-cell and delta-cell clusters. Analysis of several single-cell human pancreatic transcriptome data sets revealed 59% of GHSR-expressing cells coexpress PPY, 95% coexpress SST, and 57% coexpress PPY + SST. This expression was prominent in delta-cell and beta-cell clusters, also occurring in other clusters including gamma cells and alpha cells. GHSR expression levels were upregulated by type 2 diabetes mellitus in beta cells. In mice, plasma PP positively correlated with fat mass and with plasma levels of the endogenous GHSR antagonist/inverse agonist LEAP2. Plasma PP also elevated on LEAP2 and synthetic GHSR antagonist administration. These data suggest that in addition to delta cells, beta cells, and alpha cells, PP-expressing pancreatic cells likely represent important direct targets for LEAP2 and/or ghrelin both in mice and humans.