Nucleus accumbens ghrelin signaling controls anxiety-like behavioral response to acute stress.

BACKGROUND: Anxiety disorders are one of the most common mental disorders. Ghrelin is a critical orexigenic brain-gut peptide that regulates food intake and metabolism. Recently, the ghrelin system has attracted more attention for its crucial roles in psychiatric disorders, including depression and anxiety. However, the underlying neural mechanisms involved have not been fully investigated. METHODS: In the present study, the effect and underlying mechanism of ghrelin signaling in the nucleus accumbens (NAc) core on anxiety-like behaviors were examined in normal and acute stress rats, by using immunofluorescence, qRT-PCR, neuropharmacology, molecular manipulation and behavioral tests. RESULTS: We reported that injection of ghrelin into the NAc core caused significant anxiolytic effects. Ghrelin receptor growth hormone secretagogue receptor (GHSR) is highly localized and expressed in the NAc core neurons. Antagonism of GHSR blocked the ghrelin-induced anxiolytic effects. Moreover, molecular knockdown of GHSR induced anxiogenic effects. Furthermore, injection of ghrelin or overexpression of GHSR in the NAc core reduced acute restraint stress-induced anxiogenic effects. CONCLUSIONS: This study demonstrates that ghrelin and its receptor GHSR in the NAc core are actively involved in modulating anxiety induced by acute stress, and raises an opportunity to treat anxiety disorders by targeting ghrelin signaling system.

Visualization of the existence of growth hormone secretagogue receptor in the rat nucleus accumbens.

The potential role of the ghrelin receptor, also known as the growth hormone secretagogue receptor (GHSR), within the nucleus accumbens (NAcc) in regulating drug addiction and feeding has been documented; however, the pattern of its expression in this site remains elusive. In this study, we characterized the expression patterns of GHSR1a and 1b, two subtypes of GHSRs, within the NAcc of the rat brain by immunohistochemistry. We visually detected GHSR signals, for the first time, at the protein level in the NAcc in which they were mostly expressed in neurons including both medium spiny neurons (MSNs) and non-MSNs. Furthermore, GHSR1a was found expressed as localized near the cellular membrane or some in the cytoplasm, whereas GHSR1b expressed solely throughout the large cytoplasmic area. The existence and subcellular expression pattern of GHSRs in the NAcc identified in this study will contribute to improving our understanding about the role of GHSR-mediated neurosignaling in feeding and drug addiction.

Peptide Drug Design Using Alchemical Free Energy Calculation: An Application and Validation on Agonists of Ghrelin Receptor.

With recent large-scale applications and validations, the relative binding free energy (RBFE) calculated using alchemical free energy methods has been proven to be an accurate measure to probe the binding of small-molecule drug candidates. On the other hand, given the flexibility of peptides, it is of great interest to find out whether sufficient sampling could be achieved within the typical time scale of such calculation, and a similar level of accuracy could be reached for peptide drugs. However, the systematic evaluation of such calculations on protein-peptide systems has been less reported. Most reported studies of peptides were restricted to a limited number of data points or lacking experimental support. To demonstrate the applicability of the alchemical free energy method for protein-peptide systems in a typical real-world drug discovery project, we report an application of the thermodynamic integration (TI) method to the RBFE calculation of ghrelin receptor and its peptide agonists. Along with the calculation, the synthesis and in vitro EC50 activity of relamorelin and 17 new peptide derivatives were also reported. A cost-effective criterion to determine the data collection time was proposed for peptides in the TI simulation. The average of three TI repeats yielded a mean absolute error of 0.98 kcal/mol and Pearson's correlation coefficient (R) of 0.77 against the experimental free energy derived from the in vitro EC50 activity, showing good repeatability of the proposed method and a slightly better agreement than the results obtained from the arbitrary time frames up to 20 ns. Although it is limited by having one target and a deduced binding pose, we hope that this study can add some insights into alchemical free energy calculation of protein-peptide systems, providing theoretical assistance to the development of peptide drugs.

CRISPR/Cas9-induced LEAP2 and GHSR1a knockout mutant zebrafish displayed abnormal growth and impaired lipid metabolism.

Investigating the principles of fish fat deposition and conducting related research are current focal points in fish nutrition. This study explores the endocrine regulation of LEAP2 and GHSR1a in zebrafish by constructing mutantmodels andexamining the effects of the endocrine factors LEAP2 and its receptor GHSR1a on zebrafish growth, feeding, and liver fat deposition. Compared to the wild type (WT), the mutation of LEAP2 results in increased feeding and decreased swimming in zebrafish. The impact is more pronounced in adult female zebrafish, characterized by increased weight, length, width, and accumulation of lipid droplets in the liver.Incontrast, deficiency in GHSR1a significantly reduces the growth of male zebrafish and markedly decreases liver fat deposition.These research findings indicate the crucial roles of LEAP2 and GHSR1a in zebrafish feeding, growth, and intracellular fat metabolism. This study, for the first time, investigated the endocrine metabolic regulation functions of LEAP2 and GHSR1a in the model organism zebrafish, providing initial insights into their effects and potential mechanisms on zebrafish fat metabolism.

Chronic Social Defeat Stress Increases Brain Permeability to Ghrelin in Male Mice.

Ghrelin is a stomach-derived hormone that increases feeding and is elevated in response to chronic psychosocial stressors. The effects of ghrelin on feeding are mediated by the binding of ghrelin to the growth hormone secretagogue receptor (GHSR), a receptor located in hypothalamic and extrahypothalamic regions important for regulating food intake and metabolic rate. The ability of ghrelin to enter the brain, however, seems to be restricted to circumventricular organs like the median eminence and the brainstem area postrema, whereas ghrelin does not readily enter other GHSR-expressing regions like the ventral tegmental area (VTA). Interestingly, social stressors result in increased blood-brain barrier permeability, and this could therefore facilitate the entry of ghrelin into the brain. To investigate this, we exposed mice to social defeat stress for 21 d and then peripherally injected a Cy5-labelled biologically active ghrelin analog. The results demonstrate that chronically stressed mice exhibit higher Cy5-ghrelin fluorescence in several hypothalamic regions in addition to the ARC, including the hippocampus and midbrain. Furthermore, Cy5-ghrelin injections resulted in increased FOS expression in regions associated with the reward system in chronically stressed mice. Further histologic analyses identified a reduction in the branching of hypothalamic astrocytes in the ARC-median eminence junction, suggesting increased blood-brain barrier permeability. These data support the hypothesis that during metabolically challenging conditions like chronic stress, ghrelin may be more able to cross the blood-brain barrier and diffuse throughout the brain to target GHSR-expressing brain regions away from circumventricular organs.

GHSR Deletion in ß-Cells of Male Mice: Ineffective in Obesity, but Effective in Protecting against Streptozotocin-Induced ß-Cell Injury in Aging.

Insulin secretion from pancreatic ß cells is a key pillar of glucose homeostasis, which is impaired under obesity and aging. Growth hormone secretagogue receptor (GHSR) is the receptor of nutrient-sensing hormone ghrelin. Previously, we showed that ß-cell GHSR regulated glucose-stimulated insulin secretion (GSIS) in young mice. In the current study, we further investigated the effects of GHSR on insulin secretion in male mice under diet-induced obesity (DIO) and streptozotocin (STZ)-induced ß-cell injury in aging. ß-cell-specific-Ghsr-deficient (Ghsr-ßKO) mice exhibited no glycemic phenotype under DIO but showed significantly improved ex vivo GSIS in aging. We also detected reduced insulin sensitivity and impaired insulin secretion during aging both in vivo and ex vivo. Accordingly, there were age-related alterations in expression of glucose transporter, insulin signaling pathway, and inflammatory genes. To further determine whether GHSR deficiency affected ß-cell susceptibility to acute injury, young, middle-aged, and old Ghsr-ßKO mice were subjected to STZ. We found that middle-aged and old Ghsr-ßKO mice were protected from STZ-induced hyperglycemia and impaired insulin secretion, correlated with increased expression of insulin signaling regulators but decreased pro-inflammatory cytokines in pancreatic islets. Collectively, our findings indicate that ß-cell GHSR has a major impact on insulin secretion in aging but not obesity, and GHSR deficiency protects against STZ-induced ß-cell injury in aging.

A case control study investigating the methylation levels of GHRL and GHSR genes in alcohol use disorder.

BACKGROUND: Alcohol use disorder (AUD) is a relapsing disease described as excessive use of alcohol. Evidence of the role of DNA methylation in addiction is accumulating. Ghrelin is an important peptide known as appetite hormone and its role in addictive behavior has been identified. Here we aimed to determine the methylation levels of two crucial genes (GHRL and GHSR) in ghrelin signaling and further investigate the association between methylation ratios and plasma ghrelin levels. METHODS: Individuals diagnosed with (n = 71) and without (n = 82) AUD were recruited in this study. DNA methylation levels were measured through methylation-sensitive high-resolution melting (MS-HRM). Acylated ghrelin levels were detected by ELISA. The GHRL rs696217 polymorphism was analyzed by the standard PCR-RFLP method. RESULTS: GHRL was significantly hypermethylated (P < 0.0022) in AUD between 25 and 50% methylation than in control subjects but no significant changes of GHSR methylation were observed. Moreover, GHRL showed significant positive correlation of methylation ratio between 25 and 50% with age. A significant positive correlation between GHSR methylation and ghrelin levels in the AUD group was determined (P = 0.037). The level of GHRL methylation and the ghrelin levels showed a significant association in the control subjects (P = 0.042). CONCLUSION: GHSR and GHRL methylation levels did not change significantly between control and AUD groups. However, GHRL and GHSR methylations seemed to have associations with plasma ghrelin levels in two groups. This is the first study investigating the DNA methylation of GHRL and GHSR genes in AUD.

GHSR in a Subset of GABA Neurons Controls Food Deprivation-Induced Hyperphagia in Male Mice.

The growth hormone secretagogue receptor (GHSR), primarily known as the receptor for the hunger hormone ghrelin, potently controls food intake, yet the specific Ghsr-expressing cells mediating the orexigenic effects of this receptor remain incompletely characterized. Since Ghsr is expressed in gamma-aminobutyric acid (GABA)-producing neurons, we sought to investigate whether the selective expression of Ghsr in a subset of GABA neurons is sufficient to mediate GHSR's effects on feeding. First, we crossed mice that express a tamoxifen-dependent Cre recombinase in the subset of GABA neurons that express glutamic acid decarboxylase 2 (Gad2) enzyme (Gad2-CreER mice) with reporter mice, and found that ghrelin mainly targets a subset of Gad2-expressing neurons located in the hypothalamic arcuate nucleus (ARH) and that is predominantly segregated from Agouti-related protein (AgRP)-expressing neurons. Analysis of various single-cell RNA-sequencing datasets further corroborated that the primary subset of cells coexpressing Gad2 and Ghsr in the mouse brain are non-AgRP ARH neurons. Next, we crossed Gad2-CreER mice with reactivable GHSR-deficient mice to generate mice expressing Ghsr only in Gad2-expressing neurons (Gad2-GHSR mice). We found that ghrelin treatment induced the expression of the marker of transcriptional activation c-Fos in the ARH of Gad2-GHSR mice, yet failed to induce food intake. In contrast, food deprivation-induced refeeding was higher in Gad2-GHSR mice than in GHSR-deficient mice and similar to wild-type mice, suggesting that ghrelin-independent roles of GHSR in a subset of GABA neurons is sufficient for eliciting full compensatory hyperphagia in mice.

Ghrelin alleviates intestinal ischemia-reperfusion injury by activating the GHSR-1α/Sirt1/FOXO1 pathway.

Ischemia-reperfusion (IR) injury is primarily characterized by the restoration of blood flow perfusion and oxygen supply to ischemic tissue and organs, but it paradoxically leads to tissue injury aggravation. IR injury is a challenging pathophysiological process that is difficult to avoid clinically and frequently occurs during organ transplantation, surgery, shock resuscitation, and other processes. The major causes of IR injury include increased levels of free radicals, calcium overload, oxidative stress, and excessive inflammatory response. Ghrelin is a newly discovered brain-intestinal peptide with anti-inflammatory and antiapoptotic effects that improve blood supply. The role and mechanism of ghrelin in intestinal ischemia-reperfusion (IIR) injury remain unclear. We hypothesized that ghrelin could attenuate IIR-induced oxidative stress and apoptosis. To investigate this, we established IIR by using a non-invasive arterial clip to clamp the root of the superior mesenteric artery (SMA) in mice. Ghrelin was injected intraperitoneally at a dose of 50 µg/kg 20 min before IIR surgery, and [D-Lys3]-GHRP-6 was injected intraperitoneally at a dose of 12 nmol/kg 20 min before ghrelin injection. We mimicked the IIR process with hypoxia-reoxygenation (HR) in Caco-2 cells, which are similar to intestinal epithelial cells in structure and biochemistry. Our results showed that ghrelin inhibited IIR/HR-induced oxidative stress and apoptosis by activating GHSR-1α. Moreover, it was found that ghrelin activated the GHSR-1α/Sirt1/FOXO1 signaling pathway. We further inhibited Sirt1 and found that Sirt1 was critical for ghrelin-mediated mitigation of IIR/HR injury. Overall, our data suggest that pretreatment with ghrelin reduces oxidative stress and apoptosis to attenuate IIR/HR injury by binding with GHSR-1α to further activate Sirt1.

Genetic or pharmacological GHSR blockade has sexually dimorphic effects in rodents on a high-fat diet.

The stomach-derived hormone ghrelin regulates essential physiological functions. The ghrelin receptor (GHSR) has ligand-independent actions; therefore, GHSR gene deletion may be a reasonable approach to investigate the role of this system in feeding behaviors and diet-induced obesity (DIO). Here, we investigate the effects of a long-term (12-month) high-fat (HFD) versus regular diet on obesity-related measures in global GHSR-KO and wild-type (WT) Wistar male and female rats. Our main findings are that the GHSR gene deletion protects against DIO and decreases food intake during HFD in male but not in female rats. GHSR gene deletion increases thermogenesis and brain glucose uptake in male rats and modifies the effects of HFD on brain glucose metabolism in a sex-specific manner, as assessed with small animal positron emission tomography. We use RNA-sequencing to show that GHSR-KO rats have upregulated expression of genes responsible for fat oxidation in brown adipose tissue. Central administration of a novel GHSR inverse agonist, PF-5190457, attenuates ghrelin-induced food intake, but only in male, not in female mice. HFD-induced binge-like eating is reduced by inverse agonism in both sexes. Our results support GHSR as a promising target for new pharmacotherapies for obesity.

The allostery and modification of hGHRH molecules and specific dimer produced significant fertility effect by proliferating and activating in-situ ovarian mesenchymal stem cells.

The negative coordination of growth hormone secretagogue receptor (GHS-R) and growth hormone-releasing hormone receptor (GHRH-R) involves in the repair processes of cellular injury. The allosteric U- or H-like modified GHRH dimer Grinodin and 2Y were comparatively evaluated in normal Kunming mice and hamster infertility models induced by CPA treatment. 1-3-9 µg of Grinodin or 2Y per hamster stem-cell-exhaustion model was subcutaneously administered once a week, respectively inducing 75-69-46 or 45-13-50 % of birth rates. In comparison, the similar mole of human menopausal gonadotropin (hMG) or human growth hormone (hGH) was administered once a day but caused just 25 or 20 % of birth rates. Grinodin induced more big ovarian follicles and corpora lutea than 2Y, hMG, hGH. The hMG-treated group was observed many distorted interstitial cells and more connective tissues and the hGH-treated group had few ovarian follicles. 2Y had a plasma lifetime of 21 days and higher GH release in mice, inducing lower birth rate and stronger individual specificity in reproduction as well as only promoting the proliferation of mesenchymal-stem-cells (MSCs) in the models. In comparison, Grinodin had a plasma lifetime of 30 days and much lower GH release in mice. It significantly promoted the proliferation and activation of ovarian MSCs together with the development of follicles in the models by increasing Ki67 and GHS-R expressions, and decreasing GHRH-R expression in a dose-dependent manner. However, the high GH and excessive estrogen levels in the models showed a dose-dependent reduction in fertility. Therefore, unlike 2Y, the low dose of Grinodin specifically shows low GHS-R and high GHRH-R expressions thus evades GH and estrogen release and improves functions of organs, resulting in an increase of fertility.

Role of ghrelin hormone in the development of alcohol-associated liver disease.

Fatty liver is the earliest response of the liver to excessive alcohol consumption. Previously we identified that chronic alcohol administration increases levels of stomach-derived hormone, ghrelin, which by reducing circulating insulin levels, ultimately contributes to the development of alcohol-associated liver disease (ALD). In addition, ghrelin directly promotes fat accumulation in hepatocytes by enhancing de novo lipogenesis. Other than promoting ALD, ghrelin is known to increase alcohol craving and intake. In this study, we used a ghrelin receptor (GHSR) knockout (KO) rat model to characterize the specific contribution of ghrelin in the development of ALD with emphasis on energy homeostasis. Male Wistar wild type (WT) and GHSR-KO rats were pair-fed the Lieber-DeCarli control or ethanol diet for 6 weeks. At the end of the feeding period, glucose tolerance test was conducted, and tissue samples were collected. We observed reduced alcohol intake by GHSR-KOs compared to a previous study where WT rats were fed ethanol diet ad libitum. Further, when the WTs were pair-fed to GHSR-KOs, the KO rats exhibited resistance to develop ALD through improving insulin secretion/sensitivity to reduce adipose lipolysis and hepatic fatty acid uptake/synthesis and increase fatty acid oxidation. Furthermore, proteomic data revealed that ethanol-fed KO exhibit less alcohol-induced mitochondrial dysfunction and oxidative stress than WT rats. Proteomic data also confirmed that the ethanol-fed KOs are insulin sensitive and are resistant to hepatic steatosis development compared to WT rats. Together, these data confirm that inhibiting ghrelin action prevent alcohol-induced liver and adipose dysfunction independent of reducing alcohol intake.

Intestinal expression profiles and hepatic expression of LEAP2, ghrelin and their common receptor, GHSR, in humans.

Liver-expressed antimicrobial peptide 2 (LEAP2) and ghrelin have reciprocal effects on their common receptor, the growth hormone secretagogue receptor (GHSR). Ghrelin is considered a gastric hormone and LEAP2 a liver-derived hormone and both have been proposed to be involved in the pathophysiology of obesity and type 2 diabetes (T2D). We investigated the mRNA expression of LEAP2, ghrelin and GHSR along the intestinal tract of individuals with and without TD2, and in the liver of men with and without obesity. Mucosal biopsies retrieved with 30-cm intervals throughout the small intestine and from 7 well-defined locations along the large intestine from 12 individuals with T2D and 12 healthy controls together with liver biopsies from 15 men with obesity and 15 lean men were subjected to bulk transcriptomics analysis. Both in individuals with and without T2D, mRNA expression of LEAP2 increased through the small intestine until dropping at the ileocecal valve, with little LEAP2 mRNA expression in the large intestine. Pronounced LEAP2 expression was observed in the liver of men with and without obesity. Robust ghrelin mRNA expression was observed in the duodenum of individuals with and without T2D, gradually decreasing along the small intestine with little expression in the large intestine. Ghrelin mRNA expression was not detected in the liver biopsies, and GHSR mRNA expression was not. In conclusion, we provide unique mRNA expression profiles of LEAP2, ghrelin and GHSR along the human intestinal tract showing no T2D-associated changes, and in the liver showing no differences between men with and without obesity.

Growth Hormone Secretagogue Receptor (GHSR) Signaling in the Ventral Tegmental Area (VTA) Mediates Feeding Produced by Chronic Social Defeat Stress in Male Mice.

Ghrelin, a hormone secreted by the stomach, binds to the growth hormone secretagogue receptor (GHSR) in various brain regions to produce a number of behavioral effects that include increased feeding motivation. During social defeat stress, ghrelin levels rise in correlation with increased feeding and potentially play a role in attenuating the anxiogenic effects of social defeat. One region implicated in the feeding effects of ghrelin is the ventral tegmental area (VTA), a region implicated in reward seeking behaviors, and linked to social defeat in mice. Here we examined the role of GHSR signaling in the VTA in feeding behavior in mice exposed to social defeat stress. Male C57BL/J6 mice that were socially defeated once daily for 3 weeks ate more, had higher plasma ghrelin level and increased GHSR expression in the VTA compared to non-stressed mice. Socially defeated GHSR KO mice failed to increase their caloric intake in response to this stressor but rescue of GHSR expression in the VTA restored feeding responses. Finally, we pharmacologically blocked VTA GHSR signalling with JMV2959 infused via an indwelling VTA cannula connected to a minipump. Vehicle-treated mice increased their caloric intake during social defeat, but JMV2959-infusions attenuated feeding responses and increased anxiety-like behaviors. The data suggest that GHSR signalling in the VTA is critical for the increases in appetite observed during chronic social defeat stress. Furthermore, these data support the idea that GHSR signaling in the VTA may also have anxiolytic effects, and blocking GHSR in this region may result in an anxiety-like phenotype.

miR-128-3p Regulates Follicular Granulosa Cell Proliferation and Apoptosis by Targeting the Growth Hormone Secretagogue Receptor.

The proliferation and apoptosis of granulosa cells (GCs) affect follicle development and reproductive disorders, with microRNAs playing a crucial regulatory role. Previous studies have shown the differential expression of miR-128-3p at different stages of goat follicle development, which suggests its potential regulatory role in follicle development. In this study, through the Cell Counting Kit-8 assay, the EDU assay, flow cytometry, quantitative real-time polymerase chain reaction, Western blot, and the dual-luciferase reporter assay, we used immortal human ovarian granulosa tumor cell line (KGN) cells as materials to investigate the effects of miR-128-3p and its predicted target gene growth hormone secretagogue receptor (GHSR) on GC proliferation and apoptosis. The results show that overexpression of miR-128-3p inhibited the proliferation of KGN cells, promoted cell apoptosis, and suppressed the expression of proliferating cell nuclear antigen (PCNA) and B-cell lymphoma-2 (BCL2) while promoting that of Bcl-2 associated X protein (BAX). The dual-luciferase reporter assay revealed that miR-128-3p bound to the 3' untranslated region sequence of GHSR, which resulted in the inhibited expression of GHSR protein. Investigation of the effects of GHSR on GC proliferation and apoptosis revealed that GHSR overexpression promoted the expression of PCNA and BCL2, enhanced GC proliferation, and inhibited cell apoptosis, whereas the opposite effects were observed when GHSR expression was inhibited. In addition, miR-128-3p and GHSR can influence the expression of extracellular signal-regulated kinase 1/2 protein. In conclusion, miR-128-3p inhibits KGN cell proliferation and promotes cell apoptosis by downregulating the expression of the GHSR gene.

Neuronal ablation of GHSR mitigates diet-induced depression and memory impairment via AMPK-autophagy signaling-mediated inflammation.

Obesity is associated with chronic inflammation in the central nervous system (CNS), and neuroinflammation has been shown to have detrimental effects on mood and cognition. The growth hormone secretagogue receptor (GHSR), the biologically relevant receptor of the orexigenic hormone ghrelin, is primarily expressed in the brain. Our previous study showed that neuronal GHSR deletion prevents high-fat diet-induced obesity (DIO). Here, we investigated the effect of neuronal GHSR deletion on emotional and cognitive functions in DIO. The neuron-specific GHSR-deficient mice exhibited reduced depression and improved spatial memory compared to littermate controls under DIO. We further examined the cortex and hippocampus, the major regions regulating cognitive and emotional behaviors, and found that the neuronal deletion of GHSR reduced DIO-induced neuroinflammation by suppressing proinflammatory chemokines/cytokines and decreasing microglial activation. Furthermore, our data showed that neuronal GHSR deletion suppresses neuroinflammation by downregulating AMPK-autophagy signaling in neurons. In conclusion, our data reveal that neuronal GHSR inhibition protects against DIO-induced depressive-like behavior and spatial cognitive dysfunction, at least in part, through AMPK-autophagy signaling-mediated neuroinflammation.

Evaluating the Safety and Efficacy of Capromorelin in Rhesus Macaques (Macaca mulatta).

Nonhuman primates used in biomedical research may experience clinically significant weight loss for a variety of reasons. Episodes of anorexia (complete loss of appetite) or hyporexia (decreased appetite) can result in significant weight loss, potentially altering animal welfare and scientific studies. The FDA has approved several appetite stimulants for use in domestic species, but currently none are approved for use in NHP. Treatment of inappetence and weight loss in NHP often relies on the extralabel use of these compounds. Capromorelin is a ghrelin receptor agonist. As a growth hormone secretagogue, capromorelin increases appetite, leading to weight gain. Studies in several species have shown a positive correlation between capromorelin administration and weight gain; in 2017, an oral solution of capromorelin received FDA approval for use in dogs. We tested this solution in healthy adult rhesus macaques (n = 3 males and 3 females) for its effects on body weight and insulin like growth factor-1 (IGF-1). A control group (n = 2 males and 2 females) was used for comparison. Treated macaques received a 3mg/kg oral dose daily for 7 d. Clinical signs were observed daily. Weights were collected before, during and at the end of treatment. Blood was drawn before, during and after treatment for measurement of IGF-1 levels and standard hematology and biochemistry parameters. Baseline-adjusted mean body weights and IGF-1 levels were significantly higher in treated as compared with control monkeys after 7 d of beginning treatment (body weight of 10.5±0.1kg (mean ± SEM) and 10.1±0.1kg, respectively; IGF-1 of 758±43ng/mL and 639±22ng/mL, respectively). Capromorelin administration was not associated with appreciable changes in hematologic and biochemical values in treated macaques. These findings suggest that capromorelin may be useful for treating inappetence and weight loss in NHP, and based on blood analysis, a 7-d course of treatment does not appear to cause acute toxicity.

Identification, chemical synthesis, and receptor binding of a reptilian gecko ghrelin.

Ghrelin is known to be a gastrointestinal peptide hormone in vertebrates. It has a unique posttransrational modification, octanoylation, at the Ser side chain of the third position. In this study, we identified the genes encoding ghrelin and its receptor from the Schlegel's Japanese gecko Gekko japonicus. The C-terminal residue of gecko ghrelin was His, although the chemical synthesis method for the O-octanoyl peptide with a C-terminal His residue has not yet been well-established. Acyl-ghrelin has been synthesized using a Ser derivative without side chain protecting group in the solid-phase peptide synthesis, although this synthetic strategy has not yet been well-established. Here we show the efficient synthetic method with minimal side reactions, and G. japonicus ghrelin could be obtained in good yield. This would be useful and applicable to the synthesis of ghrelin from other animal species. The gecko ghrelin receptor was expressed in HEK 293 cells, which was fully responsive to the synthetic gecko ghrelin. These results indicate that the ghrelin system similar to mammals also exists in a reptilian gecko, G. japonicus.

Synthesis and biological evaluation of novel 18F-labeled 2,4-diaminopyrimidine derivatives for detection of ghrelin receptor in the brain.

The ghrelin receptor (GHSR) is known to regulate various physiological processes including appetite, food intake, and growth hormone release. Its expression is mainly observed in the brain, pancreas, stomach, and intestine. However, the functions of the receptor have not been fully elucidated. GHSR imaging with positron emission tomography (PET) is expected to further understanding of the functions and pathologies of the receptor. In this study, we newly designed and synthesized diaminopyrimidine derivatives ([18F]BPP-1 and [18F]BPP-2) and evaluated their utility as novel PET probes targeting GHSR. In in vitro competitive binding assays, the binding affinity of BPP-2 for GHSR (Ki = 274 nM) was comparable to that of the diaminopyimidine lead compound Abb8a (Ki = 109 nM). In a biodistribution study using normal mice, [18F]BPP-2 displayed low uptake in the brain and moderate uptake in the pancreas, but high radioactivity accumulation in bone was observed due to its defluorination in vivo. Taken together, although further improvement of the pharmacokinetics is needed, the diaminopyrimidine scaffold has potential for the development of useful GHSR-targeting PET probes.

Phytomedicine Fructus Aurantii-derived two absorbed compounds unlock antidepressant and prokinetic multi-functions via modulating 5-HT3/GHSR.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Aurantii (FA), a well-known phytomedicine, has been employed to evoke antidepressant and prokinetic multi-functions. Therein, systematically identifying bioactive components and the referred mechanism is essential for FA. AIM OF THE STUDY: This study was planned to answer "2 W" (What and Why), such as which components and pathways contribute to FA's multi-functions. We aimed to identify bioactive compounds as the key for opening the lock of FA's multi-functions, and the molecule mechanisms are their naturally matched lock cylinder. MATERIALS AND METHODS: The phytochemical content of FA extract was determined, and the compounds were identified in rats pretreated with FA using liquid chromatography with mass spectrometry (LC-MS). The contribution strategy was used to assess bioactive compounds' efficacy (doses = their content in FA) in model rats with the mechanism. The changes in functional brain regions were determined via 7.0 T functional magnetic resonance imaging-blood oxygen level-dependent (fMRI-BOLD). RESULT: Eight phytochemicals' content was detected, and merely six components were identified in rats in vivo. Meranzin hydrate + hesperidin (MH), as the primary contributor of FA, exerted antidepressant and prokinetic effects (improvement of indexes for immobility time, gastric emptying, intestinal transit, CRH, ghrelin, ACTH, DA, NA, 5-HT, CORT, and 5-HT3) by regulating 5-HT3/Growth hormone secretagogue receptor (GHSR) pathway. These results were validated by 5-HT2A, 5-HT3, and GHSR receptor antagonists combined with molecule docking. MH restored the excessive BOLD activation of the left accumbens nucleus, left corpus callosum and hypothalamus preoptic region. CONCLUSION: Absorbed MH accounts for FA's anti-depressant and prokinetic efficacy in acutely-stressed rats, primarily via 5-HT3/GHSR shared regulation.