Ghrelin Receptor Research Articles

Nutrient-sensing growth hormone secretagogue receptor in macrophage programming and meta-inflammation

ObjectiveObesity-associated chronic inflammation, aka meta-inflammation, is a key pathogenic driver for obesity-associated comorbidity. Growth hormone secretagogue receptor (GHSR) is known to mediate the effects of nutrient-sensing hormone ghrelin in food intake and fat deposition. We previously reported that global Ghsr ablation protects against diet-induced inflammation and insulin resistance, but the site(s) of action and mechanism are unknown. Macrophages are key drivers of meta-inflammation. To unravel the role of GHSR in macrophages, we generated myeloid-specific Ghsr knockout mice (LysM-Cre;Ghsrf/f). MethodsLysM-Cre;Ghsrf/f and control Ghsrf/f mice were subjected to 5 months of high-fat diet (HFD) feeding to induce obesity. In vivo, metabolic profiling of food intake, physical activity, and energy expenditure, as well as glucose and insulin tolerance tests (GTT and ITT) were performed. At termination, peritoneal macrophages (PMs), epididymal white adipose tissue (eWAT), and liver were analyzed by flow cytometry and histology. For ex vivo studies, bone marrow-derived macrophages (BMDMs) were generated from the mice and treated with palmitic acid (PA) or lipopolysaccharide (LPS). For in vitro studies, macrophage RAW264.7 cells with Ghsr overexpression or Insulin receptor substrate 2 (Irs2) knockdown were studied. ResultsWe found that Ghsr expression in PMs was increased under HFD feeding. In vivo, HFD-fed LysM-Cre;Ghsrf/f mice exhibited significantly attenuated systemic inflammation and insulin resistance without affecting food intake or body weight. Tissue analysis showed that HFD-fed LysM-Cre;Ghsrf/f mice have significantly decreased monocyte/macrophage infiltration, pro-inflammatory activation, and lipid accumulation, showing elevated lipid-associated macrophages (LAMs) in eWAT and liver. Ex vivo, Ghsr-deficient macrophages protected against PA- or LPS-induced pro-inflammatory polarization, showing reduced glycolysis, increased fatty acid oxidation, and decreased NF-κB nuclear translocation. At molecular level, GHSR metabolically programs macrophage polarization through PKA-CREB-IRS2-AKT2 signaling pathway. ConclusionsThese novel results demonstrate that macrophage GHSR plays a key role in the pathogenesis of meta-inflammation, and macrophage GHSR promotes macrophage infiltration and induces pro-inflammatory polarization. These exciting findings suggest that GHSR may serve as a novel immunotherapeutic target for the treatment of obesity and its associated comorbidity.

Effect of Anamorelin, a Ghrelin Receptor Agonist, on Muscle and Bone in Adults With Osteosarcopenia.

Anamorelin, a ghrelin receptor agonist known to stimulate the pulsatile release of GH from the pituitary, has the potential to improve musculoskeletal health in adults with osteosarcopenia. To determine the effect of anamorelin treatment for 1 year on muscle mass and strength and on biochemical markers of bone turnover in adults with osteosarcopenia (OS). Randomized, placebo-controlled, 1-year anamorelin intervention trial. The Bone Metabolism Laboratory at the USDA Nutrition Center at Tufts University. 26 men and women, age 50 years and older, with OS. Muscle mass by D3-creatine dilution and lean body mass (LBM) and bone mineral density (BMD) by dual-energy X-ray absorptiometry, muscle strength, serum IGF-1, and bone turnover markers, serum procollagen 1 intact N-terminal (P1NP), and C-terminal telopeptide (CTX). Anamorelin did not have a significant effect on muscle mass or LBM; it significantly increased knee flexion torque at 240°/s by 20% (P = .013) and had a similar nonstatistically significant effect on change in knee extension; it increased bone formation (P1NP) by 75% (P = .006) and had no significant effect on bone resorption (CTX) or BMD. Serum IGF-1 increased by 50% in the anamorelin group and did not change in the placebo group (P = .0001 for group difference). In this pilot study, anamorelin did not significantly alter muscle mass; however, it may potentially improve lower extremity strength and bone formation in addition to increasing circulating IGF-1 levels in adults with OS. Further study of anamorelin in this population is warranted.

Systemic inflammation adversely affects response to anamorelin in patients with pancreatic cancer.

Anamorelin, a selective ghrelin receptor agonist, has been approved for pancreatic cancer treatment in Japan. We aimed to investigate whether systemic inflammation, represented by the neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), and C-reactive protein (CRP)-albumin ratio (CAR), could predict the effect of anamorelin in patients with advanced pancreatic cancer. This study included 31 patients who had received anamorelin for advanced pancreatic cancer between 2021 and 2023. Patients' NLR, PLR, LMR, and CAR were evaluated before anamorelin administration. The patients were classified as responders and non-responders based on whether they gained body weight after 3months of anamorelin administration. We investigated the association between systemic inflammation and anamorelin efficacy using a univariate analysis. Twelve (39%) patients were non-responders. A high serum CRP level (p = 0.007) and high CAR (p = 0.013) was associated with non-response to anamorelin. According to the receiver operating characteristics analysis, the CAR cutoff value was 0.06, and CAR ≥ 0.06 was a risk factor (odds ratio, 5.6 [95% confidence interval 1.2-27.1], p = 0.032) for non-response to anamorelin. CAR can be a predictor of non-response to anamorelin in patients with advanced pancreatic cancer, suggesting the importance of a comprehensive assessment of the inflammatory status.

Exploring Metabolic Pathways of Anamorelin, a Selective Agonist of the Growth Hormone Secretagogue Receptor, via Molecular Networking.

In this study, we delineated the poorly characterized metabolism of anamorelin, a growth hormone secretagogue receptor agonist, in vitro using human liver microsomes (HLM), based on classical molecular networking (MN) and feature-based molecular networking (FBMN) from the Global Natural Products Social Molecular Networking platform. Following the in vitro HLM reaction, the MN analysis showed 11 neighboring nodes whose information propagated from the node corresponding to anamorelin. The FBMN analysis described the separation of six nodes that the MN analysis could not achieve. In addition, the similarity among neighboring nodes could be discerned via their respective metabolic pathways. Collectively, 18 metabolites (M1-M12) were successfully identified, suggesting that the metabolic pathways involved were demethylation, hydroxylation, dealkylation, desaturation, and N-oxidation, whereas 6 metabolites (M13a*-b*, M14a*-b*, and M15a*-b*) remained unidentified. Furthermore, the major metabolites detected in HLM, M1 and M7, were dissimilar from those observed in the CYP3A4 isozyme assay, which is recognized to be markedly inhibited by anamorelin. Specifically, M7, M8, and M9 were identified as the major metabolites in the CYP3A4 isozyme assay. Therefore, a thorough investigation of metabolism is imperative for future in vivo studies. These findings may offer prospective therapeutic opportunities for anamorelin.

Ghrelin receptor antagonism and satiety attenuate Pavlovian-instrumental transfer

Animals rely on learned cues to guide their behaviour for rewards such as food. The Pavlovian-instrumental transfer (PIT) task can be used to investigate the influence of Pavlovian stimuli on instrumental responding. Ghrelin, an orexigenic peptide, and its receptor, growth hormone secretagogue receptor 1A (GHS-R1A), has received growing interest for its role in reward-motivated learning and behaviours. A significant population of GHS-R1A have been identified within the ventral tegmental area (VTA), a critical node in the mesolimbic reward circuit that is necessary for the expression of PIT. As ghrelin has been found to increase dopaminergic activity in the VTA, we predicted that GHS-R1A antagonism with JMV-2959 would attenuate PIT. Further, given the relationship between hunger levels and changes in ghrelin signalling, we sought to compare the effects GHS-R1A antagonism with those of satiety, hypothesizing parallel effects, with each attenuating PIT. Rats received daily sessions of Pavlovian and then instrumental training over 3 weeks. Across three experiments, we examined the effects of a shift to satiety, or treatment with the GHS-R1A antagonist JMV-2959, either peripherally or directly into the VTA. We found that presentations of a stimulus paired with food reward enhanced responding for food across all conditions, thus demonstrating the expected PIT effect. Further, GHS-R1A antagonism, both peripherally and within the VTA, as well as satiety significantly reduced the magnitude of the PIT effect compared to control conditions. These results clarify our understanding of ghrelin signalling in PIT and begin to elucidate the role of feeding-related peptides in the modulation of reward-related responding.

Hypobaric hypoxia induces iron mobilization from liver and spleen and increases serum iron via activation of ghrelin/GHSR1a/MAPK signalling pathway in mice

Hypobaric hypoxia (HH) exposure affects appetite and serum iron levels in both humans and animals. Thus, whether appetite-regulating ghrelin is involved in iron regulation under HH needs to be elucidated. In vivo, C57BL/6J mice were placed in a hypobaric chamber to establish a 6000-m-high altitude exposure animal model. In vitro, mouse primary hepatocytes and peritoneal macrophages were exposed to hypoxia (1% O2) to examine the effects of ghrelin on iron-regulating proteins. HH obviously reduced the body weight of mice and significantly increased the levels of erythrocytes, and also significantly enhanced the levels of serum iron and plasma ghrelin. However, iron content in the liver and spleen was decreased, while ferroportin (Fpn) expression was increased. Moreover, ghrelin significantly induced Fpn and pERK expression in both hepatocytes and macrophages under hypoxia, which were reversed by pretreatment with growth hormone secretagogue receptor 1a (GHSR1a) antagonist or pERK inhibitor. Our findings indicated that HH leads to decreased appetite and insufficient dietary intake, which may negatively regulate the levels of ghrelin. Furthermore, GHSR1a/ERK signalling pathway is further activated to upregulate the expression of Fpn, and then promoting iron mobilization both in the liver/hepatocytes and spleen/macrophages in mice. Thus, these results revealed that ghrelin may be a potential iron regulatory hormone, and raised the possibility of ghrelin as a promising therapeutic target against iron disorders under HH.

Predictors of efficacy of anamorelin in patients with non‑small cell lung cancer and cachexia: A retrospective study.

Anamorelin, a ghrelin receptor agonist, is approved in Japan for the treatment of cachexia in patients with lung and gastrointestinal cancer. However, there is limited research on the usefulness of anamorelin in clinical settings, therefore, the present study evaluated its efficacy using patient characteristics. A total of 40 patients with non-small cell lung cancer and cachexia who were prescribed anamorelin in the Department of Respiratory Medicine, Hirosaki University Graduate School of Medicine (Aomori, Japan) between July 2021 and November 2022, were retrospectively assessed. Anamorelin was prescribed at a dose of 100 mg once daily to patients who had lost >5% of their body weight within 6 months. All patients were weighed before treatment and those who continued anamorelin treatment for 12 weeks were also weighed at 12 weeks. A logistic regression analysis was used to analyze the association between background characteristics and early discontinuation of treatment with anamorelin (within 4 weeks). The median age was 67 years (range, 36-88), and 65% of the patients were male. There were 24 patients (60.0%) with an Eastern Cooperative Oncology Group Performance Status (ECOG-PS) score 1, 11 patients (27.5%) with an ECOG-PS score 2 and five patients (12.5%) with an ECOG-PS score 3. The early discontinuation group included 11 patients (27.5%). An ECOG-PS score ≥2 (odds ratio, 7.85; 95% confidence interval, 1.43-43.21; P=0.018) was associated with early discontinuation. A total of 18/40 patients (45.0%) were able to continue anamorelin treatment for 12 weeks, and the mean change in body weight was +2.31 kg, which was a significant change from the weight recorded at baseline (P=0.027). The mean changes in lean body mass and soft lean mass between baseline and 12 weeks were +1.97 kg (P=0.14) and +1.26 kg (P=0.15), respectively. The results from the present study indicate that anamorelin is unlikely to be useful for patients with a poor general condition (ECOG-PS score ≥2).

From the stomach to locus coeruleus: new neural substrate for ghrelin's effects on ingestive, motivated and anxiety-like behaviors.

Ghrelin, a stomach-derived orexigenic hormone, has a well-established role in energy homeostasis, food reward, and emotionality. Noradrenergic neurons of the locus coeruleus (LC) are known to play an important role in arousal, emotion, cognition, but recently have also been implicated in control of feeding behavior. Ghrelin receptors (the growth hormone secretagogue receptor, GHSR) may be found in the LC, but the behavioral effects of ghrelin signaling in this area are still unexplored. Here, we first determined whether GHSR are present in the rat LC, and demonstrate that GHSR are expressed on noradrenergic neurons in both sexes. We next investigated whether ghrelin controls ingestive and motivated behaviors as well as anxiety-like behavior by acting in the LC. To pursue this idea, we examined the effects of LC GHSR stimulation and blockade on food intake, operant responding for a palatable food reward and, anxiety-like behavior in the open field (OF) and acoustic startle response (ASR) tests in male and female rats. Our results demonstrate that intra-LC ghrelin administration increases chow intake and motivated behavior for sucrose in both sexes. Additionally, females, but not males, exhibited a potent anxiolytic response in the ASR. In order to determine whether activation of GHSR in the LC was necessary for feeding and anxiety behavior control, we utilized liver-expressed antimicrobial peptide 2 (LEAP2), a newly identified endogenous GHSR antagonist. LEAP2 delivered specifically into the LC was sufficient to reduce fasting-induced chow hyperphagia in both sexes, but food reward only in females. Moreover, blockade of GHSR in the LC increased anxiety-like behavior measured in the ASR test in both sexes. Taken together, these results indicate that ghrelin acts in the LC to alter ingestive, motivated and anxiety-like behaviors, with a degree of sex divergence.

Relamorelin in Gastroparesis and Diabetic Gastroparesis: A Meta-Analysis on Its Efficacy and Safety.

This meta-analysis aimed to evaluate the efficacy and safety of relamorelin, a synthetic ghrelin receptor agonist, for the treatment of gastroparesis and diabetic gastroparesis. A total of 1,033 participants from five randomized controlled trials were included. The primary outcome was the mean change in gastric emptying time from baseline. Relamorelin demonstrated a statistically significant improvement in gastric emptying time with a mean difference of -11.40 minutes compared to the placebo group. Furthermore, a significant improvement was observed specifically in diabetic gastroparesis patients, with a mean difference of -8.43 minutes. However, adverse effects, such as headaches, dizziness, and gastrointestinal symptoms, were more prevalent in the relamorelin group. Despite these adverse effects, the study concludes that relamorelin offers a promising avenue for gastroparesis treatment, especially given the limited existing therapeutic options. This comprehensive meta-analysis synthesizes existing evidence to inform clinical practice and guides future research in this domain.

Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model.

Adiponectin (AdipoQ) is an adipokine involved in glucose homeostasis and lipid metabolism. In mammals, its role in appetite control is highly controversial. To shed light on the comparative aspects of AdipoQ in lower vertebrates, goldfish was used as a model to study feeding regulation by AdipoQ in fish species. As a first step, goldfish AdipoQ was cloned and found to be ubiquitously expressed at the tissue level. Using sequence alignment, protein modeling, phylogenetic analysis and comparative synteny, goldfish AdipoQ was shown to be evolutionarily related to its fish counterparts and structurally comparable with AdipoQ in higher vertebrates. In our study, recombinant goldfish AdipoQ was expressed in E. coli, purified by IMAC, and confirmed to be bioactive via activation of AdipoQ receptors expressed in HepG2 cells. Feeding in goldfish revealed that plasma levels of AdipoQ and its transcript expression in the liver and brain areas involved in appetite control including the telencephalon, optic tectum, and hypothalamus could be elevated by food intake. In parallel studies, IP and ICV injection of recombinant goldfish AdipoQ in goldfish was effective in reducing foraging behaviors and food consumption. Meanwhile, transcript expression of orexigenic factors (NPY, AgRP, orexin, and apelin) was suppressed with parallel rises in anorexigenic factors (POMC, CART, CCK, and MCH) in the telencephalon, optic tectum and/or hypothalamus. In these brain areas, transcript signals for leptin receptor were upregulated with concurrent drops in the NPY receptor and ghrelin receptors. In the experiment with IP injection of AdipoQ, transcript expression of leptin was also elevated with a parallel drop in ghrelin mRNA in the liver. These findings suggest that AdipoQ can act as a novel satiety factor in goldfish. In this case, AdipoQ signals (both central and peripheral) can be induced by feeding and act within the brain to inhibit feeding behaviors and food intake via differential regulation of orexigenic/anorexigenic factors and their receptors. The feeding inhibition observed may also involve the hepatic action of AdipoQ by modulation of feeding regulators expressed in the liver.

Ghrelin signalling in AgRP neurons links metabolic state to the sensory regulation of AgRP neural activity

ObjectiveThe sensory detection of food and food cues suppresses Agouti related peptide (AgRP) neuronal activity prior to consumption with greatest suppression occurring in response to highly caloric food or interoceptive energy need. However, the interoceptive mechanisms priming an appropriate AgRP neural response to external sensory information of food availability remain unexplored. Since hunger increases plasma ghrelin, we hypothesized that ghrelin receptor (GHSR) signalling on AgRP neurons is a key interoceptive mechanism integrating energy need with external sensory cues predicting caloric availability. MethodsWe used in vivo photometry to measure the effects of ghrelin administration or fasting on AgRP neural activity with GCaMP6s and dopamine release in the nucleus accumbens with GRAB-DA in mice lacking ghrelin receptors in AgRP neurons. ResultsThe deletion of GHSR on AgRP neurons prevented ghrelin-induced food intake, motivation and AgRP activity. The presentation of food (peanut butter pellet) or a wooden dowel suppressed AgRP activity in fasted WT but not mice lacking GHSRs in AgRP neurons. Similarly, peanut butter and a wooden dowel increased dopamine release in the nucleus accumbens after ip ghrelin injection in WT but not mice lacking GHSRs in AgRP neurons. No difference in dopamine release was observed in fasted mice. Finally, ip ghrelin administration did not directly increase dopamine neural activity in the ventral tegmental area. ConclusionsOur results suggest that AgRP GHSRs integrate an interoceptive state of energy need with external sensory information to produce an optimal change in AgRP neural activity. Thus, ghrelin signalling on AgRP neurons is more than just a feedback signal to increase AgRP activity during hunger.

New insights into the molecular basis of alcohol abstinence and relapse in alcohol-associated liver disease.

Alcohol use disorder (AUD) remains a significant public health concern, affecting around 5% of adults worldwide. Novel pathways of damage have been described during the last years, providing insight into the mechanism of injury due to alcohol misuse beyond the direct effect of ethanol byproducts on the liver parenchyma and neurobehavioral mechanisms. Thus, the gut-liver-brain axis and immune system involvement could be therapeutic targets for AUD. In particular, a change in gut microbiota composition and function, especially bile acid homeostasis, and these changes can improve after alcohol cessation. Alcohol can also directly disrupt intestinal and blood-brain barriers. Activation of the immune system can be triggered by intestinal barrier dysfunction and translocation of bacteria, pathogen-associated molecular patterns (such as lipopolysaccharide), cytokines, and damage-associated molecular patterns. These factors in turn promote liver and brain inflammation and progression of liver fibrosis. Other involved mechanisms include oxidative stress, apoptosis, autophagy, and the release of extracellular vesicles and miRNA from hepatocytes. Potential therapeutic targets include gut microbiota (probiotics and fecal microbiota transplantation), neuroinflammatory pathways, as well as neuroendocrine pathways, e.g.: the ghrelin system (ghrelin receptor blockade), incretin mimetics (GLP-1 analogs), and the mineralocorticoid receptor system (spironolactone). In addition, support with psychological and behavioral treatments is essential to address the multiple dimensions of AUD. In the future, a personalized approach considering these novel targets can contribute to significantly decreasing the alcohol-related burden of disease.

Reduction of compulsive overeating in rats caused by maternal deprivation in early ontogenesis with the use of a new ghrelin receptor antagonist agrelax

BACKGROUND: Factors that may trigger episodes of binge eating include mental and physical stress, dietary restrictions of high-calorie foods. In a rodent model it has been shown that intermittent consumption of high-calorie foods causes binge eating regardless of body weight gain.
 AIM: To investigate the effect of a novel ghrelin receptor antagonist Agrelax on binge eating in in adult rats after maternal deprivation in early ontogeny.
 MATERIALS AND METHODS: Animals were weaned for 180 min from day 2 to day 12 after birth; males 90100 days of age were used in the experiments. In the development of binge eating, animals received a high-carbohydrate diet (Nutella paste based chocolate mixture) for 1 h every day or every third day for 1.5 months. Fifteen minutes before feeding, the chocolate paste was placed within 5 cm of reach with visual contact. Agrelax, a novel ghrelin receptor antagonist, was administered intranasally 1g/1l, 20l for 7 days.
 RESULTS: Maternal deprivation induced bindge eating of high-calorie foods in adult rats. When chocolate was given 3 times a week, its consumption increased (p 0.001) in the maternal deprivation group relative to the control group. After a course of administration of agrelax, chocolate consumption did not differ significantly from that in the control group. The daily consumption of standard food did not differ relative to the control group both before and after the course of agrelax administration. When chocolate was given daily, the maternal deprivation rats did not develop food addiction. At the same time agrelax did not induce a change in chocolate consumption relative to the control group.
 CONCLUSIONS: The findings suggest new ways to synthesize pharmacological agents of peptide nature based on ghrelin and its antagonists for correction of food addiction caused by psychogenic stresses in ontogenesis.

OR12-04 LEAP2 Deletion Does Not Counteract The Effects Of Ghrelin Deletion During Insulin-Induced-Hypoglycemia

Abstract Disclosure: K. Shankar: None. S. Varshney: None. D. Gupta: None. O. Singh: None. S.B. Ogden: None. S. Osborne-Lawrence: None. N.P. Metzger: None. C.P. Richard: None. J.M. Zigman: Grant Recipient; Self; Novo Nordisk. Objective: The hormones ghrelin and LEAP2 both are endogenous ligands for the growth hormone secretagogue receptor (GHSR). Whereas ghrelin activates GHSR, LEAP2 blocks ghrelin from activating GHSR and also inhibits GHSR constitutive activity. It is via GHSR that the glucoregulatory actions of ghrelin and LEAP2 are mediated. These glucoregulatory actions are highlighted by the findings of severe hypoglycemia in ghrelin-KO mice and LEAP2-overexpressing mice when they are submitted to an acute-on-chronic caloric restriction protocol. Also, recently, our group showed that ghrelin-KO mice require a much higher glucose infusion rate (GIR) and exhibit markedly reduced elevation of counterregulatory hormones when submitted to a hyperinsulinemic-hypoglycemic clamp. In the current study, we investigated the potential of LEAP2 to protect against insulin-induced-hypoglycemia and counteract the effects of ghrelin deletion during the hypoglycemic clamp. Methods: Eight-ten week-old male wild-type (WT), Ghrelin-KO, LEAP2-KO, and LEAP2/Ghrelin-doubleKO (lacking both ghrelin and LEAP2 ) littermates were used for this study (n=7-10 per genotype). Mice were implanted with a right jugular vein catheter. Five days later, hyperinsulinemic-hypoglycemic clamps were performed in conscious, unrestrained mice. A low dose of insulin was infused at 4 mU/kg/min i.v. over 2 hr. A 20% glucose solution was simultaneously infused at a variable rate to achieve hypoglycemia (35–45 mg/dL) during the final 30 min. Blood glucose was measured via tail nicks every 5 min. Results: We were able to achieve the target hypoglycemic range in all four genotypes using this protocol. As compared to WT mice, Ghrelin-KO mice required markedly elevated (∼110% higher) GIRs (10±4 mg/kg/min in WT vs. 22±2 mg/kg/min in Ghrelin-KO mice; p=0.03) by the end of the 2-hr clamp. The GIRs required by LEAP2-KO mice were equivalent to those of WT mice (7±2 mg/kg/min in LEAP2-KO; p=n.s.). Furthermore, although during the first 60 min of the clamp, LEAP2/Ghrelin-doubleKO mice exhibited slightly lower blood glucose levels than the other genotypes and during the middle 60 min of the clamp, they required higher GIRs than the other genotypes, by the end of the 2-hr clamp, their GIRs were similar to Ghrelin-KO mice (25±3 mg/Kg/min in Ghrelin/LEAP2-doubleKO mice, p=n.s.). Conclusions: These data suggest a complex effect of LEAP2 on blood glucose. Alone, LEAP2 deletion does not impact insulin sensitivity. When coupled with ghrelin deletion, LEAP2 deletion lowers fasting blood glucose and causes greater insulin sensitivity early on during the hyperinsulinemic-hypoglycemic clamp. By the end of the clamp, this effect of LEAP2 deletion when coupled to ghrelin deletion is gone. Altogether, these results suggest that LEAP2 deletion does not counteract the effects of ghrelin deletion during insulin-induced hypoglycemia. Presentation: Friday, June 16, 2023

OR21-06 Growth Response Of Oral LUM-201 In OraGrowtH210 And OraGrowtH212 Trials In Idiopathic Pediatric Growth Hormone Deficiency (iPGHD): Combined Analysis Interim Analysis Data

Abstract Disclosure: M.J. Tansey: Research Investigator; Self; Lumos Pharma, Inc. S.A. Bowden: Research Investigator; Self; Lumos Pharma, Inc. A.N. Dauber: Research Investigator; Self; Lumos Pharma, Inc. B. Wikiera: Research Investigator; Self; Lumos Pharma, Inc. B. Pyrzak: Research Investigator; Self; Lumos Pharma, Inc. A.T. Bossowski: Research Investigator; Self; Lumos Pharma, Inc. E. Petriczko: Research Investigator; Self; Lumos Pharma, Inc. R. Stawerska: Research Investigator; Self; Lumos Pharma, Inc. E. Moszczynska: Research Investigator; Self; Lumos Pharma, Inc. F. Cassorla: Research Investigator; Self; Lumos Pharma, Inc. Speaker; Self; Lumos Pharma, Inc. M.M. Feldt: Research Investigator; Self; Lumos Pharma, Inc. A.J. Lunsford: Research Investigator; Self; Lumos Pharma, Inc. M.E. Gottschalk: Research Investigator; Self; Lumos Pharma, Inc. M. Marin: Research Investigator; Self; Lumos Pharma, Inc. S.N. Nayak: Research Investigator; Self; Lumos Pharma, Inc. S. Bhuvana: Research Investigator; Self; Lumos Pharma, Inc. D.R. Repaske: Research Investigator; Self; Lumos Pharma, Inc. L.A. Soyka: Research Investigator; Self; Lumos Pharma, Inc. J.S. Fuqua: Research Investigator; Self; Lumos Pharma, Inc. O. Escobar: Research Investigator; Self; Lumos Pharma, Inc. D.A. Bowlby: Research Investigator; Self; Lumos Pharma, Inc. P.Y. Fechner: Research Investigator; Self; Lumos Pharma, Inc. E. Wiltshire: Research Investigator; Self; Lumos Pharma, Inc. M. Harris: Research Investigator; Self; Lumos Pharma, Inc. K.A. Wintergerst: Research Investigator; Self; Lumos Pharma, Inc. A.R. Lafferty: Research Investigator; Self; Lumos Pharma, Inc. B.S. Miller: Research Investigator; Self; Lumos Pharma, Inc. P. Simm: Research Investigator; Self; Lumos Pharma, Inc. A. Bruchey: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. C. Smith: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. D.B. Karpf: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. J.C. McKew: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. M.O. Thorner: Consulting Fee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. Background: LUM-201 (ibutamoren), a growth hormone (GH) secretagogue receptor 1a (GHSR1a) agonist, is a potent, long-acting investigational oral GH secretagogue currently studied in three Idiopathic Pediatric GH Deficiency (iPGHD) studies. The LUM-201 predictive enrichment marker (PEM) is used to identify patients diagnosed with iPGHD (peak stimulated GH >3<10 ng/mL) who are likely to respond to LUM-201. PEM positivity is defined as a baseline insulin-like growth factor-1 (IGF-1) level >30 ng/mL and a peak GH of ≥5 ng/mL in response to a single 0.8 mg/kg dose of LUM-201. Objectives: Report the growth response analyzing the combined interim analysis (IA) data from two Phase 2 trials studying LUM-201 at two different doses (1.6 mg/kg/day or 3.2 mg/kg/day). Methods: IA data from both studies were combined and analyzed for calculated annualized height velocity (AHV). Baseline demographics were analyzed for the two combined cohorts. Results: After 6 months of treatment with LUM-201, the calculated AHV (mean ±SD ) was 8.1±1.9 cm/year in the 1.6 mg/kg/day group and 8.0±1.5 cm/year in the 3.2 mg/kg/day group (N=15 in both groups). After 9 months of treatment, the calculated AHV was 7.8±1.7 cm/year in the 1.6 mg/kg/day group and 7.3±1.7 cm/year in the 3.2 mg/kg/day group (N=10 in both groups). After 12 months of treatment, the calculated AHV was 7.8±1.7 cm/year in the 1.6 mg/kg/day group and 7.4 ±1.2 cm/year in the 3.2 mg/kg/day group (N=6 in both groups). LUM-201 was well tolerated; no safety concerns were identified across the dose range in adverse events (AE) data, laboratory values, and ECG values. Conclusions: As the growth velocity was comparable for the two doses of oral LUM-201, this analysis of the combined IA data appears to strongly support 1.6 mg/kg/day as the optimal dose for the Phase 3 trial, as doubling the dose appeared to offer no meaningful improvement in efficacy. Final determination will await final full data set analysis of both studies. Presentation: Saturday, June 17, 2023

OR0303 Syndecans Enhance Ghrelin-Induced GHSR1a Signaling

Abstract Disclosure: K. Prins: None. M. Huisman: None. R. Mies: None. P.J. Delhanty: None. J.A. Visser: None. Ghrelin is a gut hormone that enhances food intake and growth hormone secretion through its receptor, the G-protein coupled receptor (GPCR) GHSR1a. Recently, we showed that ghrelin interacts with syndecans (SDCs), a family of four membrane proteins that have been linked to obesity in genome-wide association studies. Here, we investigated whether SDCs impact ghrelin signaling at GHSR1a. We used HEK293 cells that expressed GHSR1a and SDC1, SDC2, SDC3, or SDC4 through transient transfection. GHSR1a mainly signals through the Gαq pathway, so we first explored the effects of SDCs on ghrelin-induced intracellular Ca2+ influx. Using a mitochondrial-targeted aequorin, this Ca2+ response can be measured seconds after ghrelin stimulation. We found that all SDCs increased the maximum Ca2+ response at least 4.2-fold (P<0.001), without affecting the EC50. This enhanced Ca2+ response was not caused by increased receptor availability: SDCs reduced cell membrane receptor expression by 51-85% (P<0.001). This low GHSR1a membrane availability contrasted with the increased Ca2+ response, and raised the question whether SDCs could be an alternate receptor for ghrelin. To test this, we transfected cells with SDC1 alone, but this did not permit a ghrelin-stimulated Ca2+ response, indicating that the ghrelin-induced Ca2+ response was GHSR1a-dependent. Moreover, the SDC-potentiated response was also Gαq-dependent, since GNAQ knockout cells transfected with GHSR1a and SDC1 expression plasmids failed to generate a Ca2+ response. However, using a Gq biosensor, we found that the level of activation of Gαq by ghrelin was not affected by SDC1 transfection, suggesting an effect of SDC1 either downstream of Gαq or on the quenching of GHSR. Thus, we investigated the interaction between GHSR1a and β-arrestin 2. Β-arrestin 2 downregulates GHSR function following activation by sterically hindering G-protein coupling and mediating receptor internalization. Four minutes after ghrelin stimulation, SDCs reduced β-arrestin 2 recruitment by 69-87% (P<0.001). SDC-transfected cells also reached a maximum β-arrestin 2 response 8-14 minutes later than GHSR1a only-transfected cells. Notably, these differences in β-arrestin 2 recruitment occurred minutes after the Ca2+ response. These findings suggest that the reduction in β-arrestin 2 recruitment does not cause the increased Ca2+ response in the presence of SDCs, and that the effect of SDCs is probably downstream of Gαq. Interestingly, the effects of SDCs mimic those of melanocortin receptor accessory protein 2 (MRAP2). MRAP2 also enhances the ghrelin-stimulated activation of the Gαq signaling pathway, independently of β-arrestin recruitment. Altogether, we showed that SDCs enhanced Ca2+ influx and reduced β-arrestin 2 recruitment to GHSR1a in response to ghrelin. This could be a novel mechanism through which SDCs affect metabolism and obesity. Presentation: Thursday, June 15, 2023

OR21-03 Dose Responsiveness Of LUM-201 As Measured By Acute GH Response And IGF-1 And Annualized Height Velocity (AHV) Measured At 6 Months In The Interim Analysis Of The OraGrowtH212 Study In Idiopathic Pediatric Growth Hormone Deficiency (iPGHD)

Abstract Disclosure: F. Cassorla: Research Investigator; Self; Lumos Pharma, Inc. Speaker; Self; Lumos Pharma, Inc. R. Román: Research Investigator; Self; Lumos Pharma, Inc. M.L. Johnson: Consulting Fee; Self; Lumos Pharma, Inc. A. Avila: Research Investigator; Self; Lumos Pharma, Inc. G. Iñiguez: Research Investigator; Self; Lumos Pharma, Inc. I. Baier: Research Investigator; Self; Lumos Pharma, Inc. D. Said: Research Investigator; Self; Lumos Pharma, Inc. A. Bruchey: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. C. Smith: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. E.L. Brincks: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. J.C. McKew: None. D.B. Karpf: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. M.O. Thorner: Consulting Fee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. Objective: To evaluate acute GH, IGF-1, AHV at 6 mos of treatment of iPGHD subjects. Background: LUM-201 is an investigational oral GH secretagogue (GHS) currently in 3 Phase 2 iPGHD trials. The LUM-201 predictive enrichment marker (PEM) may be used to identify patients previously diagnosed with iPGHD who are likely to respond to LUM-201. PEM positivity is defined as a baseline IGF-1 level >30 ng/mL and a peak GH of ≥5 ng/mL in response to a single 0.8 mg/kg LUM-201 dose. LUM-201, a potent, durable oral GHS agonist, acts on the GHS Receptor 1a in the hypothalamus and anterior pituitary. LUM-201 enhances the amplitude of endogenous GH pulsatile release over 24 hrs, while also acting as a functional antagonist of the somatostatin receptor. The increased IGF-1 and GH reach the growth plates and promote linear growth. Three advantages of LUM-201 as a potential therapeutic for iPGHD are that it is a small oral tablet, it enhances natural GH pulses over 24 hr, and GH release is subject to the endogenous negative feedback mechanisms, so serum IGF-1 is expected to remain within the normal range. Method: Each subject is screened with the PEM test, which includes determining the max GH concentration in the first 90 min after a single 0.8 mg/kg dose of LUM-201. Additionally, each of these PEM positive subjects was assessed for increasing doses of LUM-201 to induce further acute GH secretion on their first day of therapeutic dosing with either a 1.6 or 3.2 mg/kg dose. The first 10 subjects with 6 months of growth on LUM-201 (5 subjects on each dose) were evaluated on their IGF-1 levels and AHVs. Results: The mean (+SD, n) peak GH for the 0.8 mg/kg dose was 26.04 ng/mL (+10.6, 22), at the 1.6 mg/kg dose 35.50 ng/mL (+17.5, 9), and at a 3.2 mg/kg dose 38.34 ng/mL (+10.4, 8). Serum IGF-1 levels of the 5 subjects in each cohort were monitored at baseline and at 6 months of treatment. The mean (+ SD) serum IGF-1 levels showed a change from baseline of 84.6 ng/mL (+30.1) at 6 months for the 1.6 mg/kg cohort and 113.8 (+49.9) at the 3.2 mg/kg dose. The pretreatment HV were 4.9 (+ 0.4) at 1.6 mg/kg and 4.3 (+ 0.9) cm/year at 3.2 mg/kg respectively and the respective mean AHVs (+ SD) at 6 months were 7.14 cm/year (+0.65) and 8.60 cm/year (+1.22). The acute GH response demonstrated a dose response between the 0.8 and 1.6 mg/kg doses, but the 1.6 and 3.2 mg/kg doses produced similar GH responses. These two doses of LUM-201 induced comparable changes from baseline values of IGF-1 after 6 months of treatment. In addition to these pharmacodynamic changes, the AHVs generated from each dose also appear similar. Conclusion: LUM-201 generated the expected AHV in this naive iPGHD population. The dose-response for GH secretion after an initial dose of LUM-201 and increases from baseline of serum IGF-1 after 6 months of treatment suggest that 1.6 mg/kg/day is the optimum dose for efficacy and durability. The results from this small study are corroborated by the larger OraGrowtH210 study data. Presentation: Saturday, June 17, 2023

DNA Methylation Testing for Endometrial Cancer Detection in Urine, Cervicovaginal Self-Samples and Cervical Scrapes

ABSTRACT Endometrial cancer is the sixth most common cancer in women globally and has a rising incidence, accounting for 417,000 new diagnoses and more than 97,000 deaths in 2020. Early detection is critical because of a poor advanced-stage prognosis and high relapse risk. Postmenopausal bleeding precedes endometrial cancer in 90% of cases, yet only 5% to 10% of patients with this symptom have an underlying malignancy. Recent cytology research has demonstrated endometrial cancer cells in vaginal samples, suggesting urine and cervicovaginal self-samples may allow convenient and minimally invasive screening. In addition, screening samples for DNA methylation in tumor suppressor genes can increase testing efficacy of triaging patients with postmenopausal bleeding as it does not require intact tumor cells. This study aimed to evaluate the diagnostic performance of endometrial cancer detection using DNA methylation analysis in paired urine, cervicovaginal self-samples, and clinician-taken cervical scrapes. Paired samples from participants of the SOLUTION1 study with endometrial cancer were collected between October 2016 and August 2020. A complete urine void and cervicovaginal self-sample was collected at home, whereas the cervical scrape was taken by a clinician in the operating room before surgery. Each patient had a paired urine, cervicovaginal self-sample, and clinician-taken cervical scrape available for methylation analysis. Controls were collected from the Urine Controls (URIC) biobank and Dutch national cervical cancer screening program. Promoter hypermethylation of the ADCYAP1, BHLHE22, CDH13, CDO1, GALR1, GHSR, HAND2, SST, and ZIC1 genes was tested using quantitative methylation-specific polymerase chain reaction. Optimal 3-marker combinations were determined for each sample type using multivariable logistic regression analysis, and diagnostic performances of each marker and 3-marker panels were assessed by leave-one-out cross validation. A total of 103 endometrial cancer patients were included in this study, with 317 unpaired samples of control women. Methylation levels of all markers were significantly higher in all 3 sample types of endometrial cancer patients compared with healthy control women. In urine, the non–cross-validated area under the curve of the DNA methylation markers ranged between 0.61 and 0.93, in cervicovaginal self-samples between 0.62 and 0.91, and in clinician-taken cervical scrapes between 0.61 and 0.95. Most markers (7/9) showed the highest performance in urine, with the remaining 2 showing best performance in clinician-taken cervical scrapes. The optimal 3-marker combination panels showed area under the curve values of 0.95 (05% confidence interval [CI], 0.92–0.98) for urine, 0.94 (95% CI, 0.90–0.97) for cervicovaginal self-samples, and 0.97 (95% CI, 0.96–0.99) for clinician-taken cervical scrapes. The sensitivity and specificity of the optimal urine testing panel were both 90%, were 89% sensitivity and 92% specificity for cervicovaginal self-samples, and were 93% sensitivity and 90% specificity for clinician-taken cervical scrapes. The leave-one-out cross-validation analysis revealed virtually equal performances in marker panels for each of the 3 testing modalities. The results of this study demonstrate high diagnostic potential of DNA methylation testing in minimally and noninvasive samples for endometrial cancer detection, emphasizing the potential for patient-friendly home-based sample collection.

Differential impact of a ghrelin receptor antagonist or inverse agonist in the electrical kindling model of epilepsy

Ghrelin is a peptide, which has been shown to affect seizures. However, there is not a consensus about its real impact on the control of seizure severity. We assessed the influence of intra-amygdala injections of a ghrelin receptor (GHSR) antagonist, as well as a GHSR inverse agonist on the electrical kindling-induced seizures. Two unipolar electrodes and a tripolar electrode twisted with a guide cannula were implanted in the skull surface or the basolateral amygdala of adult male rats, respectively. A rapid electrical kindling protocol was applied for kindling epileptogenesis. The stimulations were applied until rats showed three consecutive stage five seizures. Each rat was considered as its control. D-Lys-3-GHRP-6 (1, 12.5, and 25 μg/rat) or [D-Arg, D-phe, D-Trp, heu] substance P (D-SP) (50, 500 and 5000 ng/rat) as the GHSR antagonist or inverse agonist were injected into the basolateral amygdala. Seizure parameters including after-discharge duration (ADD), stage five duration (S5D), and seizure stage (SS) were documented thirty minutes following administration of the drugs or saline. Antagonism of the GHSR in the amygdala, significantly increased seizure induction in the kindled rats, in a dose-dependent manner, and induced spontaneous seizures leading to status epilepticus. Conversely, D-SP had a dose-dependent anticonvulsant activity, indicated by decreased ADD and S5D. The results show that GHSR inverse agonism suppressed seizure severity in the rat amygdala kindling model, whereas GHSR antagonism made seizures more severe. Therefore, when considering the ghrelin system to modulate seizures, it is crucial to note the differential impact of various GHSR ligands.

Sites and mechanisms of action of colokinetics at dopamine, ghrelin and serotonin receptors in the rodent lumbosacral defecation centre.

Agonists of dopamine D2 receptors (D2R), 5-hydroxytryptamine (5-HT, serotonin) receptors (5-HTR) and ghrelin receptors (GHSR) activate neurons in the lumbosacral defecation centre, and act as 'colokinetics', leading to increased propulsive colonic motility, in vivo. In the present study, we investigated which neurons in the lumbosacral defecation centre express the receptors and whether dopamine, serotonin and ghrelin receptor agonists act on the same lumbosacral preganglionic neurons (PGNs). We used whole cell electrophysiology to record responses from neurons in the lumbosacral defecation centre, following colokinetic application, and investigated their expression profiles and the chemistries of their neural inputs. Fluorescence in situ hybridisation revealed Drd2, Ghsr and Htr2C transcripts were colocalised in lumbosacral PGNs of mice, and immunohistochemistry showed that these neurons have closely associated tyrosine hydroxylase and 5-HT boutons. Previous studies showed that they do not receive ghrelin inputs. Whole cell electrophysiology in adult mice spinal cord revealed that dopamine, serotonin, α-methylserotonin and capromorelin each caused inward, excitatory currents in overlapping populations of lumbosacral PGNs. Furthermore, dopamine caused increased frequency of both IPSCs and EPSCs in a cohort of D2R neurons. Tetrodotoxin blocked the IPSCs and EPSCs, revealing a post-synaptic excitatory action of dopamine. In lumbosacral PGNs of postnatal day 7-14 rats, only dopamine's postsynaptic effects were observed. Furthermore, inward, excitatory currents evoked by dopamine were reduced by the GHSR antagonist, YIL781. We conclude that lumbosacral PGNs are the site where the action of endogenous ligands of D2R and 5-HT2R converge, and that GHSR act as a cis-modulator of D2R expressed by the same neurons. KEY POINTS: Dopamine, 5-hydroxytryptamine (5-HT, serotonin) and ghrelin (GHSR) receptor agonists increase colorectal motility and have been postulated to act at receptors on parasympathetic preganglionic neurons (PGNs) in the lumbosacral spinal cord. We aimed to determine which neurons in the lumbosacral spinal cord express dopamine, serotonin and GHSR receptors, their neural inputs, and whether agonists at these receptors excite them. We show that dopamine, serotonin and ghrelin receptor transcripts are contained in the same PGNs and that these neurons have closely associated tyrosine hydroxylase and serotonin boutons. Whole cell electrophysiology revealed that dopamine, serotonin and GHSR receptor agonists induce an inward excitatory current in overlapping populations of lumbosacral PGNs. Dopamine-induced excitation was reversed by GHSR antagonism. The present study demonstrates that lumbosacral PGNs are the site at which actions of endogenous ligands of dopamine D2 receptors and 5-HT type 2 receptors converge. Ghrelin receptors are functional, but their role appears to be as modulators of dopamine effects at D2 receptors.