Ghrelin Antagonist Research Articles

A new ghrelin receptor antagonist, agrelax, reduces emotional overeating caused by the stimulation of the lateral hypothalamus reward zone in well-fed rats

BACKGROUND: Ghrelin receptor antagonists hold promise for the treatment of eating disorders. The reward zone of the lateral hypothalamus has been proposed as a target for mediating the effects of the ghrelin system in emotional overeating.
 AIM: This study aimed to evaluate the effects of a new ghrelin receptor antagonist, agrelax, on emotional overeating induced by the stimulation of the reward zone of the lateral hypothalamus in well-fed rats.
 MATERIALS AND METHODS: Male Wistar rats were trained to self-stimulate in a Skinner box. After training, a feeder was placed in the Skinner box, and a food-conditioned reflex was developed in the rats for 5 days. Then, the reaction of food self-deprivation, i.e., behavior under conditions of choice of self-stimulation or food intake, was assessed.
 RESULTS: The reaction of food self-deprivation when the animals did not approach the feeder was 10% of the threshold current. Self-stimulation of the lateral hypothalamus with a threshold current caused numerous approaches to the feeder and food intake. Sulpiride, a dopamine D2/D3 antagonist (5 and 20 mg/kg ip), administered to well-fed rats reduced both feeding behavior and the intensity of self-stimulation in the food self-deprivation test at threshold currents. The ghrelin receptor antagonists [D-LYS3]-GHRP-6 and the novel antagonist agrelax (1 µg/µL, 20 µL intranasally) also reduced both feeding behavior and the intensity of self-stimulation under these conditions.
 CONCLUSIONS: Ghrelin and dopamine receptors are involved in emotional overeating. Agrelax, a novel ghrelin receptor antagonist, reduces emotional overeating induced by the activation of the lateral hypothalamic reward system. Because emotional overeating is strongly associated with clinical eating disorders such as bulimia and binge-eating disorder, the use of ghrelin antagonists to treat and prevent this problem is promising.

SAT049 Goldfish LEAP2: Molecular Cloning, Functional Characterization, And Novel Regulation By IGF And Insulin At The Hepatic Level

Abstract Disclosure: F. Jin: None. X. Qin: None. C. Ye: None. M. He: None. A.O. Wong: None. Liver-expressed antimicrobial peptide 2 (LEAP2) is a peptide highly expressed in the liver and its secretion induced by bacterial infection constitutes an integral component of innate immunity in vertebrate species. Recently, LEAP2 has been confirmed to be the endogenous antagonist of the feeding regulator ghrelin, implying that the peptide may play a role in appetite control and energy homeostasis. However, whether LEAP2 can be regulated by metabolic/endocrine signals is still unclear and the signal transduction involved is totally unknown. Using goldfish as a model, three isoforms of LEAP2, namely LEAP2a, 2b & 2c, have been cloned and phylogenetic analysis confirmed that they could be clustered into the clade of fish LEAP2. Comparative synteny also revealed that the three isoforms were derived from separated genes with similar structural organization but located in different chromosomes of goldfish genome. The a.a. sequences and 3D protein structure of LEAP2a-c were found to be highly comparable with LEAP2 of other vertebrates and tissue expression profiling by RT-PCR also showed that they were expressed at high levels in the liver but to a lower extent in other tissues. As members of antimicrobial peptides, goldfish LEAP2a-c not only could exhibit antimicrobial activity against Staphylococcus epidermidis but also play important role in fish innate immunity, which is evidenced by their transcript expression induced by LPS & cytokines at hepatic level as well as in immune tissues in goldfish naturally infected with Aeromonas hydrophila. In goldfish liver cells, transcript expression of LEAP2a-c could be up-regulated in a time- and dose-dependent manner by IGF-I, IGF-II and insulin. Using a pharmacological approach, the stimulatory effects of IGF-I/-II and insulin on the three isoforms of LEAP2 were confirmed to be mediated by IGF-I receptor (IGF1R) but not insulin receptor activation. Furthermore, the PI3K/Akt, MEK/ERK and P38 MAPK pathways were shown to be involved in IGF-I induced LEAP2a-c gene expression. Our findings for the first time demonstrate that the endocrine signals targeting the liver (insulin) or produced locally within the liver (IGF-I & -II) can play a role in regulating LEAP2 expression at the hepatic level, which may shed light on a novel aspect of the functional crosstalk between innate immunity and feeding control/energy balance in fish model. Presentation: Saturday, June 17, 2023

The role of dysregulated ghrelin/LEAP-2 balance in anorexia nervosa

LEAP-2 is a ghrelin antagonist with an anorexigenic drive. This study investigates the evolution of plasma ghrelin and LEAP-2 concentrations in 29 patients with anorexia nervosa (AN) before and after refeeding and compares it to physiological adaptations during fasting in healthy controls or to mouse model of chronic food restriction and refeeding. Acute and chronic food restriction decrease LEAP-2 and increase ghrelin concentrations in both humans and mice, while patients with AN displayed higher ghrelin and LEAP-2 concentrations before than after refeeding (p= 0.043). After 6months follow-up, patients with unstable weight gain (n= 17) had significantly decreased LEAP-2 concentrations after refeeding (p= 0.044), in contrast to patients with stable weight gain (n= 12). We provide evidence that the ghrelin/LEAP-2 system is not regulated according to the nutritional status in AN, in contrast to what is physiologically expected when coping with food restriction.

Postprandial Increases in Liver-Gut Hormone LEAP2 Correlate with Attenuated Eating Behavior in Adults Without Obesity.

The novel liver-gut hormone liver-expressed antimicrobial peptide-2 (LEAP2) is a centrally acting inverse agonist, and competitive antagonist of orexigenic acyl ghrelin (AG), at the GH secretagogue receptor, reducing food intake in rodents. In humans, the effects of LEAP2 on eating behavior and mechanisms behind the postprandial increase in LEAP2 are unclear, though this is reciprocal to the postprandial decrease in plasma AG. Plasma LEAP2 was measured in a secondary analysis of a previous study. Twenty-two adults without obesity attended after an overnight fast, consuming a 730-kcal meal without or with subcutaneous AG administration. Postprandial changes in plasma LEAP2 were correlated with postprandial changes in appetite, high-energy (HE) or low-energy (LE) food cue reactivity using functional magnetic resonance imaging, ad libitum food intake, and plasma/serum AG, glucose, insulin, and triglycerides. Postprandial plasma LEAP2 increased by 24.5% to 52.2% at 70 to 150 minutes, but was unchanged by exogenous AG administration. Postprandial increases in LEAP2 correlated positively with postprandial decreases in appetite, and cue reactivity to HE/LE and HE food in anteroposterior cingulate cortex, paracingulate cortex, frontal pole, and middle frontal gyrus, with similar trend for food intake. Postprandial increases in LEAP2 correlated negatively with body mass index, but did not correlate positively with increases in glucose, insulin, or triglycerides, nor decreases in AG. These correlational findings are consistent with a role for postprandial increases in plasma LEAP2 in suppressing human eating behavior in adults without obesity. Postprandial increases in plasma LEAP2 are unrelated to changes in plasma AG and the mediator(s) remain uncertain.

Ghrelin levels in different brain regions in <i>Danio rerio</i> exposured to stress

BACKGROUND: Literature data indicated that ghrelin may be a stress neuropeptide in some animals, including Danio rerio.
 AIM: The aim of the study was to measure the levels of ghrelin in Danio rerio brain (forebrain, midbrain, and hindbrain) after predator exposure (predator stress test) and administration of ghrelin and oxytocin antagonists to assess the role of ghrelin in stress response.
 MATERIALS AND METHODS: The study used 68 Danio rerio, one predator of Cichlasoma nicaraguensis. Ghrelin levels were measured by enzyme-linked immunosorbent assay. The following pharmacological agents were used: 1) ghrelin antagonist hexapeptide [D-Lys3]-GHRP-6 (Tocris, UK); 2) recombinant peptide analog of ghrelin agrelax with a molecular weight of 3.5 kDa, developed at the IEM (both compounds in equal dosages of 0.333 mg/L); corticoliberin (Tocris) at a dosage of 0.4 mg/L; and oxytocin (Gedeon Richter, Hungary) at a dosage of 3.8 L (0.005 IU/L) per 50 mL of aquarium water (0.019 IU/L).
 RESULTS: In the control group, ghrelin level was determined only in the hindbrain, and ghrelin levels in the forebrain and midbrain were 4 pg/mg of total protein. Contact with a predator led to a significant increase in ghrelin levels in the forebrain and midbrain, but not in the hindbrain. Thus, in fish forebrain, ghrelin levels increased to 966 12 pg/mg of protein, which is almost a 250-fold increase in the indicator compared with the level in intact animals. In the presence of agrelax, [D-Lys3]-GHRP-6, and oxytocin, ghrelin levels in the forebrain and midbrain of stressed fish decreased approximately equally to 88 3, 97 1, and 115 1 pg/mg protein, respectively.
 CONCLUSION: Thus, stress exposure (contact with a predator) significantly increases ghrelin levels in the forebrain and midbrain but decreases in the hindbrain. Stress-protective peptides (oxytocin, [D-Lys3]-GHRP-6, and agrelax) decreased ghrelin levels in the forebrain and midbrain, and to a lesser extent, in the hindbrain when exposed to stress.

The role of dysregulated ghrelin/LEAP-2 balance in eating disorder: a translational study in anorexia nervosa.

AbstractThe ghrelin system is a key regulator of appetite and food intake across species. LEAP-2, a recently discovered ghrelin antagonist, appears to be up-regulated in obesity and opposes to the orexigenic drive of ghrelin. The evolution of LEAP-2 levels could be an interesting insight to reflect the regulation of appetite in eating disorders such as anorexia nervosa (AN). We provide the first study exploring the ghrelin and LEAP-2 regulation in long-term food restriction followed by refeeding in both mice and patients suffering from AN.Using a translational strategy, we compared the regulation of ghrelin and LEAP-2 concentrations in blood during food restriction and after refeeding in female mice exposed to a 14 days protocol combining quantitative food restriction and running wheel activity followed by progressive refeeding. We compared these results to clinical data fom in an ongoing longitudinal study of patients with AN evaluated before and after refeeding as well as 6 months after hospital discharge.Long-term food restriction in mice was associated with increased ghrelin and decreased LEAP-2 concentrations compared to ad libitum fed controls. Refeeding led to an increase in LEAP-2 concentrations. Patients with AN displayed increased ghrelin levels but also higher LEAP-2 concentrations on admission than after refeeding. LEAP-2 decreased with refeeding. On 17 patients re-evaluated 6 months after discharge, patients with unstable weight gain exhibited a greater decrease of LEAP-2 concentrations during refeeding compared to patient with stable weight gain. Decreasing LEAP-2 concentrations was able to predict a negative outcome (i.e. unstable weight gain) in 80% of the cases.We provide evidence that the ghrelin/LEAP-2 system is not regulated according to the nutritional status in AN as it is in the case of a physiological adaptation to food restriction. Our clinical data suggest that the evolution of LEAP-2 concentrations during refeeding is opposed to data from preclinical model and could give new insights on the outcome of weight gain in AN.Disclosure of InterestNone Declared

The role of dysregulated ghrelin/LEAP-2 balance in eating disorder: a translational study in anorexia nervosa

IntroductionAnorexia nervosa (AN) is a complex psychiatric disorder characterized by a persistant decrease in food intake leading to dramatic weight loss and energy deficit. The ghrelin system is a key regulator of appetite and food intake across species. LEAP-2, a recently discovered ghrelin antagonist, appears to be up-regulated in obesity and opposes to the orexigenic drive of ghrelin. The evolution of LEAP-2 levels could be an interesting insight to reflect the regulation of appetite in eating disorders such as anorexia nervosa (AN).ObjectivesWe tested this hypothesis and here provide the first study exploring the ghrelin and LEAP-2 regulation in long-term food restriction followed by refeeding in both mice and patients suffering from AN.MethodsUsing a translational strategy, we compared the regulation of ghrelin and LEAP-2 concentrations in blood during food restriction and after refeeding i/ in female mice exposed to a 14 days protocol combining quantitative food restriction and running wheel activity followed by 10 days of progressive refeeding; ii/ in an ongoing longitudinal study of patients with AN evaluated before and after refeeding (n=30) as well as 6 months after hospital discharge to evaluate if the weight gain was stable (n=7) or unstable (n=10). Plasma concentrations of ghrelin and LEAP-2 were measured with selective immunoassays.ResultsLong-term food restriction in mice was associated with increased ghrelin (p<0.001) and decreased LEAP-2 concentrations (p=0.006) compared to ad libitum fed controls. Refeeding led to a decrease in ghrelin (p<0.01) and increase in LEAP-2 concentrations (p<0.01). Patients with AN displayed increased ghrelin levels (p<0.01) but also higher LEAP-2 concentrations on admission than after refeeding (p=0.04). LEAP-2 decreased with refeeding. On 17 patients re-evaluated 6 months after discharge, patients with unstable weight gain exhibited a greater decrease of LEAP-2 concentrations during refeeding compared to patient with stable weight gain (p=0.02). Decreasing LEAP-2 concentrations was able to predict a negative outcome (i.e. unstable weight gain) in 80% of the cases.ConclusionsWe provide evidence that the ghrelin/LEAP-2 system is not regulated according to the nutritional status in AN as it is in the case of a physiological adaptation to food restriction. Results from an ongoing longitudinal study exploring remission in AN suggest that the evolution of LEAP-2 concentrations during refeeding is opposed to data from preclinical model and could give new insights on the outcome of weight gain in AN.Disclosure of InterestNone Declared

Liver-expressed antimicrobial peptide 2 is associated with improved pancreatic insulin secretion in adults with overweight and obesity.

To examine association of liver-expressed antimicrobial peptide 2 (LEAP2), an endogenous ghrelin antagonist with anorexiant effects, to key cardiometabolic risk factors in people with overweight and obesity. In this cross-sectional study, we sought to identify associations between LEAP2 levels and cardiometabolic risk factors, including body composition (dual X-ray absorptiometry), insulin and glucose metabolism (oral and intravenous glucose tolerance tests and hyperinsulinaemic-euglycaemic clamps), plasma lipids and inflammation markers (ELISA and multiplex assays). In 65 participants with overweight or obesity (63.1% male, mean age 31.3 ± 8.5 years), LEAP2 levels were associated with total body fat, but not with body mass index or waist-hip ratio in both univariable and age- and sex-adjusted models (P < 0.05). Higher LEAP2 level was also positively associated with higher insulin secretion in univariable (P=0.047) and multivariable models adjusted for age, sex and body fat (P=0.03), but not with fasting glucose levels (P ≥ 0.05). Higher LEAP2 levels were associated insulin resistance (P=0.07) after adjustment for age and sex, but the association disappeared after an additional adjustment for body fat (P=0.2). There was an inverse association between LEAP2 levels and nuclear factor kappa-B (NFκB) activity in the peripheral blood mononuclear cells in age-, sex- and body fat-adjusted models (P=0.04). There were no associations with cardiovascular risk factors (lipids, blood pressure) or other inflammation markers. These results provide important insights into the association between LEAP2 and cardiometabolic health in a high-risk population of individuals with overweight and obesity. This is a first report of an association between LEAP2 and insulin secretion, insulin sensitivity and NFκB activity. LEAP2 may represent an important potential therapeutic target to promote insulin secretion in people with type 2 diabetes and obesity.

Increased levels of plasma neudesin in adult growth hormone deficiency and their relationship with plasma liver-expressed antimicrobial peptide-2 levels: a cross-sectional study.

Adult growth hormone deficiency (aGHD) is characterized by an altered metabolic profile and increased cardiovascular risk. Neudesin is a newly discovered protein mainly secreted from adipose tissue and brain, under evaluation for its possible activity as a negative regulator of energy expenditure. Liver-expressed antimicrobial peptide (LEAP)-2 is a competitive antagonist of ghrelin on its receptor. An observational cross-sectional study was performed to test the hypothesis that plasma neudesin levels may be modified in aGHD. Given the role played in the energy balance, any possible relationships between neudesin, LEAP-2 and metabolic and anthropometric parameters were evaluated. Thirty-eight patients were included: 18 aGHD patients (7 females and 11 males, aged 59.7 ± 2.6years, BMI 30.2 ± 2.2kg/m2); 20 healthy controls (12 females and 8 males, aged 47.1 ± 2.5years, BMI 24.1 ± 0.9kg/m2). All patients were evaluated for glucose, insulin, HOMA and QUICKI index, total/LDL/HDL cholesterol, triglycerides, uric acid, and IGF-1. Plasma neudesin, LEAP-2, and ghrelin were measured by ELISA. Fat mass was evaluated by DEXA. Neudesin levels were significantly higher in aGHD versus controls. We confirmed the finding of significantly lower ghrelin levels and significantly higher LEAP-2/ghrelin ratio in aGHD patients and found a significant direct correlation between neudesin and LEAP-2 levels. A significant direct correlation between neudesin and fat mass percentage was found in the whole population. These results suggest the onset of adaptive responses to an altered metabolic picture in aGHD. The changes in two distinct pathways that modulate food intake and the still limited knowledge about neudesin suggest future developments in this field.

The changes of protein kinases activities in the brain structures after ghrelin antagonists administration in previously stressed &lt;i&gt;Danio rerio&lt;/i&gt;

BACKGROUND: Nowadays there are a lot of data to suggest about an important role of ghrelin signaling system in stress response and reinforcement mechanisms. There is a common opinion that a protein kinase system plays a role of a second intracellular messenger for ghrelin system.&#x0D; AIM: So, the aim of our study was to measure the total protein kinase activity in brain structures of Danio rerio. The all procedures have been approved according to the Local Ethical Committee of Institute of Experimental Medicine.&#x0D; MATERIALS AND METHODS: In our study it has been used 50 specimen Danio rerio 68 months of age. There also has been used a predator (Hypsophrys nicaraguensis). During experiment a fish has been firstly placed into a tank of water with a dissolved pharmacological substance for 5 min, then has been transferred into a tank with predator also for 5 min. Two ghrelin antagonists have been used in our experiment in 0.333 mg/l dosage [D-Lys3]-GHRP-6 and Agrelax, a recombinant peptide analogue of ghrelin with molecular weight 3.5 kD, which has been invented in our Institute. The brain has been divided into three anatomical parts: telencephalon, midbrain and cerebellum. After that the material for kinase activity assay was made (ADP sensor Universal Kinase Activity Assay kit, BioVision).We also have used GraphPad Prism 6 for statistical estimation. KolmogorovSmirnov test MannWhitney U-test have been used as statistical tests.&#x0D; RESULTS: In control group, the kinase activity has been determined in all tested brain structures. After predator exposure, the kinase activity has significantly changed. After [D-Lys3]-GHRP-6 administration there has not been determined any protein kinase activity in all brain structures. In the cerebellum the kinase activity has been the highest after Agrelax administration. Protein kinase system demonstrates significant stress response.&#x0D; CONCLUSIONS. Pharmacological action on ghrelin receptors leads to changes in kinase system in Danio rerios brain structures.

Meranzin Hydrate Improves Depression-Like Behaviors and Hypomotility via Ghrelin and Neurocircuitry.

To investigate whether meranzin hydrate (MH) can alleviate depression-like behavior and hypomotility similar to Chaihu Shugan Powder (CSP), and further explore the potential common mechanisms. Totally 120 Spraque-Dawley rats were randomly divided into 5-8 groups including sham, vehicle, fluoxetine (20 mg/kg), mosapride (10 mg/kg), CSP (30 g/kg), MH (9.18 mg/kg), [D-Lys3]-GHRP-6 (Dlys, 0.5 mg/kg), and MH+Dlys groups by a random number table, 8 rats in each group. And 32 mice were randomly divided into wild-type, MH (18 mg/kg), growth hormone secretagogue receptor-knockout (GHSR-KO), and GHSR+MH groups, 8 mice in each group. The forced swimming test (FST), open field test (OFT), tail suspension test (TST), gastric emptying (GE) test, and intestinal transit (IT) test were used to assess antidepressant and prokinetic (AP) effects after drug single administration for 30 min with absorbable identification in rats and mice, respectively. The protein expression levels of brain-derived neurotrophic factor (BDNF) and phosphorylated mammalian target of rapamycin (p-mTOR) in the hippocampus of rats were evaluated by Western blot. The differences in functional brain changes were determined via 7.0 T functional magnetic resonance imaging-blood oxygen level-dependent (fMRI-BOLD). MH treatment improved depression-like behavior (FST, OFT) and hypomotility (GE, IT) in the acute forced swimming (FS) rats (all P<0.05), and the effects are similar to the parent formula CSP. The ghrelin antagonist [D-Lys3]-GHRP-6 inhibited the effect of MH on FST and GE (P<0.05). Similarly, MH treatment also alleviated depression-like behavior (FST, TST) in the wild-type mice, however, no effects were found in the GHSR KO mice. Additionally, administration of MH significantly stimulated BDNF and p-mTOR protein expressions in the hippocampus (both P<0.01), which were also prevented by [D-Lys3]-GHRP-6 (P<0.01). Besides, 3 main BOLD foci following acute FS rats implicated activity in hippocampus-thalamus-basal ganglia (HTB) circuits. The [D-Lys3]-GHRP-6 synchronously inhibited BOLD HTB foci. As expected, prokinetic mosapride only had effects on the thalamus and basal ganglia, but not on the hippocampus. Within the HTB, the hippocampus is implicated in depression and FD. MH accounts for part of AP effects of parent formula CSP in acute FS rats, mainly via ghrelin-related shared regulation coupled to BOLD signals in brain areas. This novel functionally connection of HTB following acute stress, treatment, and regulation highlights anti-depression unified theory.

Ghrelin-Ghrelin receptor (GSHR) pathway via endocannabinoid signal affects the expression of NPY to promote the food intake of Siberian sturgeon (Acipenser baerii)

Previous data suggested that activation of endocannabinoid receptor 1 (CB1) was necessary for the orexigenic effect of Ghrelin in rodents, but the information is limited in teleosts. To investigate the feeding regulation pathway of Ghrelin and CB1 in Siberian sturgeon (Acipenser baerii), this study first identified the Ghrelin (345 bp, complete coding sequence) and Ghrelin receptor (GHSR, 500 bp, partial coding sequence) sequences, and then detected their tissue distribution patterns, which showed that Ghrelin is mainly distribution in peripheral tissues, while GSHR is mainly in different brain divisions. Besides, the qPCR before and after feeding showed that the mRNA expressions of Ghrelin and GHSR were inhibited after feeding in telencephalon, diencephalon and mesencephalon. Subsequently, the food intake and appetite factor expressions were measured by i.c.v. co-injection of Ghrelin and GSHR antagonist. The results showed that Ghrelin promoted the food intake of Siberian sturgeon, which was reversed by its receptor antagonist. Besides, i.c.v. injection of Ghrelin decreased telencephalon CART expression while increased NPY expression in the three brain regions. In addition, to further explore the relationship of Ghrelin and CB1 signal regulating feeding, the co-injection of Ghrelin and CB1 antagonists was performed. The results showed that AM6545 (CB1 peripheral restricted antagonist) failed to affect the orexigenic effect of Ghrelin and the expression pattern of NPY mRNA in the telencephalon. While in the diencephalon, the increase of food intake and NPY mRNA expression induced by Ghrelin was completely reversed by Rimonabant (CB1 global antagonist). These results indicate Ghrelin-GSHR pathway promotes the food intake of Siberian sturgeon by inducing the expression of NPY in the diencephalon, and the stimulating effect will be reversed by cannabinoid receptor antagonism. This study provides a foundation for understanding the pathways Ghrelin and CB1 signals in appetite regulation of the teleost.

Energetic demands of lactation produce an increase in the expression of growth hormone secretagogue receptor in the hypothalamus and ventral tegmental area of the rat despite a reduction in circulating ghrelin.

Lactating rats show changes in the secretion of hormones and brain signals that promote hyperphagia and facilitate the production of milk. Little is known, however, about the role of ghrelin in the mechanisms sustaining lactational hyperphagia. Here, we used Wistar female rats that underwent surgery to sever the galactophores to prevent milk delivery (GC rats) and decrease the energetic drain of milk delivery. We compared plasma acyl-ghrelin concentrations and growth hormone secretagogue receptor (GHSR) mRNA expression in different brain regions of GC rats with those of sham operated lactating and nonlactating rats. Additional lactating and nonlactating rats were implanted with cannulae aimed at the lateral ventricles and were used to compare feeding responses to central ghrelin or GHSR antagonist infusions to those of nonlactating rats receiving similar infusions on day 14-16 postpartum (pp). Results show lower plasma acyl-ghrelin concentrations on day 15 pp sham operated lactating rats compared to GC or nonlactating rats. These changes occur in association with increased GHSR mRNA expression in the hypothalamic arcuate nucleus (ARC) and ventral tegmental area (VTA) of sham operated lactating rats. Despite lactational hyperphagia, infusions of ghrelin (0.25 or 1μg) resulted in similar increases in food intake in lactating and nonlactating rats. In addition, infusions of the GHSR antagonist JMV3002 (4μg in 1μl of vehicle) produced greater suppression of food intake in lactating rats than in nonlactating rats. These data suggest that, despite lower plasma ghrelin, the energetic drain of lactation increases sensitivity to the orexigenic effects of ghrelin in brain regions important for food intake and energy balance, and these events are associated with lactational hyperphagia.

Effect of feeding regimen on circadian activity rhythms of food anticipatory by ghrelin hormone in a pig model

ABSTRACT Increasingly diverse meal patterns affect the internal body clock. Ghrelin secretion is closely associated with the anticipation of a regularly scheduled mealtime, leading ghrelin to be a putative candidate for food-related entraining signals that drive activity rhythms. Here, growing pigs with different meal frequencies were used to construct an irregular eating pattern model. We found that irregular eating patterns changed central ghrelin levels of pigs, affected the circadian entrainment and circadian rhythm pathways in hypothalamus tissue, and altered the daily behavior and food anticipatory activity (FAA). To determine whether ghrelin exerts an effect, growing pigs were intravenously injected with ghrelin antagonist [D-Lys3]-GHRP-6 for 7 days. We showed here that [D-Lys3]-GHRP-6 administration decreased locomotor activity of growing pigs in the 4-h window preceding onset of food availability. In addition, we also confirmed that the direct role of ghrelin in molecular mechanism of regulating clock genes expression via calcium mobilization through intracellular PKC/PLC and AC/PKA pathways in vitro. Collectively, irregular eating patterns affect the central circadian system by ghrelin, supporting ghrelin as a temporal messenger of food-entrainment in hypothalamic circadian functions.

The Ghrelin Antagonist [D-Lys3]-GHRP-6 Decreases Signs of Risk Behavior in a Model of Gambling Addiction in Rats by Altering Dopamine and Serotonin Metabolism

The Ghrelin Antagonist [D-Lys3]-GHRP-6 Decreases Signs of Risk Behavior in a Model of Gambling Addiction in Rats by Altering Dopamine and Serotonin Metabolism

Role of Dopaminergic and Cannabinoidergic Receptors on Ghrelin-Induced Hypophagia in 5-Day-Old Broiler Chicken.

The present study aimed to identify the role of dopaminergic and cannabinoidergic systems in the ghrelin-induced hypophagia among meat-type chickens. In the first experiment, intracerebroventricular (ICV) injection was applied to birds with control solution, D1 receptor antagonist (5 nmol), ghrelin (6 nmol), and D1 receptor antagonist plus ghrelin. The second to sixth experiments were similar to the first one, with the difference that D2 receptor antagonist (5 nmol), D3 receptor antagonist (6.4 nmol), D4 receptor antagonist (6 nmol), the precursor of dopamine (125 nmol), and 6-hydroxy dopamine (150 nmol) instead of D1 antagonist were injected into the broiler chickens. In experiment 7, control solution and different levels of ghrelin antagonists (5, 10, and 20 nmol) were injected. In experiment 8, the chickens were ICV injected with control solution, ghrelin antagonist (10 nmol), dopamine (40 nmol), and ghrelin antagonist plus dopamine. In experiments 9 and 10, CB1 and CB2 receptors antagonist (6.25µg and 5µg) were co-injected with ghrelin (6 nmol), respectively, measuring the food intake for 120 min after the injection. It was observed that ghrelin ICV injection considerably reduced food intake, whereas ghrelin antagonist increased food intake, depending on the dose (P<0.05). In addition, ghrelin-induced hypophagia was significantly attenuated by D1 receptor antagonist and 6-hydroxy dopamine (P<0.05), while the dopamine precursor considerably elevated the ghrelin-induced food intake (P<0.05). The dopamine-induced feeding behavior was diminished by the co-administration of [D-Lys-3]-GHRP-6 (10 nmol)+dopamine (40 nmol) (P<0.05). In addition, CB1 receptor antagonists enhanced the ghrelin influence on food intake (P<0.05). The results implied that the hypophagic impact of ghrelin was probably mediated by D1 and CB1 receptors within neonatal broilers.

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.

Ghrelin antagonist D-Lys3-GHRP-6 counteract ghrelin effects in bovine cumulus-oocytes complexes matured in vitro.

Ghrelin is a gut hormone related to energy balance and reproductive functions. The aim of this study was to evaluate the effect of ghrelin antagonist D-Lys3-GHRP-6 (GA) as a potential agent that prevents ghrelin effects during bovine oocyte maturation on progesterone production, cumulus cell (CC) viability, CC DNA damage and embryo development and hatching rates. Ghrelin's potential to induce oxidative stress in cumulus-oocyte complexes (COC) was also evaluated. COCs were cultured for 24hr in medium without supplementation (C) or supplemented with 60pM ghrelin (Ghrelin60), Ghrelin60+20pM GA (GA20), Ghrelin60+60pM GA (GA60) or Ghrelin60+100pM GA (GA100) for experiment I. For experiment II, C and Ghrelin60 treatments were used. Differences between C and Ghrelin60 and the linear or quadratic association between GAs on Ghrelin60 were evaluated. Results demonstrated that Ghrelin60 increased progesterone concentration, reduced CC viability, induced CC DNA damage and decreased blastocyst and hatching rate compared with C (p<.05). GA20, GA60 and GA100 had a linear effect on CC genetic damage index (p≤.05) and a quadratic effect on CC viability (p<.01). GA20 counteracted the low hatching rate produced by Ghrelin60. However, GAs did not counteract progesterone concentration and blastocyst rate (p≥.21). GRH60 did not differ from C in the oxidative status (p≥.19). Our study highlights that GA could prevent the negative effects of ghrelin during bovine IVM.

Circulating LEAP-2 Levels in Adult Growth Hormone Deficiency and Their Relationship With IGF-1

Liver enriched antimicrobial peptide-2 (LEAP-2) is an endogenous antagonist of ghrelin, which acts as an allosteric modulator of growth hormone secretagogue receptors. It is expressed predominantly in liver, followed by kidney, jejunum, duodenum and stomach. Its expression in conditions of metabolic impairment, obesity for instance, may be upregulated, usually pairing with a concomitant reduction in ghrelin secretion. Weight gain and hyperglycaemia seem to be the main trigger factors for LEAP-2 production. Adult growth hormone deficiency (aGHD) is known as a pathological condition characterized by metabolic impairment (insulin resistance, weight gain, increased fat mass, decreased lean mass). To the best of our knowledge, no study in literature deals with the problem of circulating LEAP-2 levels in GHD. Therefore, the primary endpoint of this cross-sectional observational study was to evaluate circulating LEAP-2 levels in GHD in comparison to healthy controls whether the secondary endpoint was to evaluate any possible correlation with IGF-1 and metabolic parameters in such condition.30 patients were included in the study. Group A included adult GHD: 15 patients, 7 females and 8 males, mean±standard error of the mean (SEM) age 54.6±3.51 years, BMI 29,95±1.63 kg/m2). The etiologies of GHD were empty sella (n=6), idiopathic (n=6), post-surgical hypopituitarism (n=2), pineal cyst (n=1). Group B was formed by controls: 15 patients, 11 females and 4 males, mean±SEM age 40.33±2.61 years, BMI 24.19±1.33 kg/m2. They were evaluated for: serum glucose and insulin, HOMA-index, QUICKI-index, Total/LDL/HDL cholesterol, triglycerides, IGF-1 and LEAP-2 (measured using Human LEAP-2 ELISA kit, Phoenix Pharmaceuticals Inc, according to manufacturer’s instructions).Circulating LEAP-2 is significantly higher in GHD than in control group (26.98±3.12 vs 18.7±1.9 ng/ml, p=0.03). LEAP-2 levels in our cohort was not influenced by BMI, while a significant direct correlation between LEAP-2 and age was detected in aGHD group. A strong significative inverse correlation between LEAP-2 and IGF-1 was evidenced in aGHD (r2=0.5). Finally, in this group, LEAP-2 inversely correlates with total cholesterol (r2=0.45).As expected circulating LEAP-2 levels, in a condition of metabolic impairment such as GHD, were higher than controls even if no correlation with BMI was evidenced in our cohort. The inverse correlation between LEAP-2 and IGF-1 in GHD patients may suggest a body adjustment in a worse clinical condition. LEAP-2 may act as a brake for ghrelin production, thus preventing further weight gain and fat mass increase, although losing its secretagogue effect.

In search of new brain biomarkers of stress

The aim: of the study was to investigate the level of ghrelin in various brain structures during a stress response in Zebrafish to a predator, to evaluate this indicator as a potential biomarker of stress, and the effect of a benzodiazepine tranquilizer (phenazepam) on stress-induced changes&#x0D; Materials and methods: The object of the study was Zebrafish, or Danio rerio wild type, which was subjected to stress by exposure to a predator Hypsophrys nicaraguensis from the cichlid family. In the tail tissue, the level of cortisol was determined, in the brain – the level of total (acylated and non-acylated) ghrelin by the method of enzyme-linked immunosorbent assay. The benzodiazepine anxiolytic phenazepam (1 mg/L), a ghrelin antagonist [D-Lys3]-GHRP-6 (0.333 mg/l) and corticotropin-releasing hormone (CRF; 0.4 mg/L) were used as the pharmacological agents.&#x0D; Results and discussion: Exposure to a predator, just as administering CRF, more than doubled the level of cortisol in the tail tissue. [D-Lys3]-GHRP-6 and phenazepam prevented an increase in a tissue cortisol level. Simultaneously, in the medulla oblongata and cerebellum, the phylogenetically most ancient structures, rather than in the forebrain (telencephalon) or in the midbrain (corpora bigemia), the level of ghrelin was recorded about 500 pg/g of total protein. In response to exposure to a predator, the level of ghrelin increased in the forebrain and midbrain to nanogram concentrations and moderately decreased in the cerebellum. The effect was prevented by phenazepam and [D-Lys3]-GHRP-6.&#x0D; Conclusion: Increases in ghrelin in the brain in response to stressful situations can be seen as a functional brain biomarker of stress, along with increased levels of tissue cortisol levels. Both of these effects are prevented by both the ghrelin antagonist and the benzodiazepine tranquilizer. The mechanism of action of the tranquilizer is a functional antagonism between the GABAergic system of the brain and the ghrelin system.