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.

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.

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.

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.

Ghrelin/GHSR signaling in the lateral septum ameliorates chronic stress-induced depressive-like behaviors.

Ghrelin is a gastrointestinal hormone on feeding and metabolism regulation, and acts through its receptor-growth hormone secretagogue receptor (GHSR), which is widely distributed throughout the central nervous system. Recent studies have suggested that ghrelin plays an important role in the regulation of depression, but the underlying mechanisms remain uncertain. Lateral septum (LS) is a critical brain region in modulating depression. Therefore, we investigated the role of ghrelin/GHSR signaling in the LS on the depressive-like behaviors of mice under conditions of chronic stress by using behavioral tests, neuropharmacology, and molecular biology techniques. We found that infusion of ghrelin into the LS produced antidepressant-like responses in mice. Activation of LS GABAergic neurons was involved in the antidepressant effect of ghrelin. Importantly, GHSR was highly expressed and distributed in the LS neurons. Blockade of GHSR in the LS reversed the ghrelin-induced antidepressant-like effects. Molecular knockdown of GHSR in the LS induced depressive-like symptoms in mice. Furthermore, administration of ghrelin into the LS alleviated depressive-like behaviors induced by chronic social defeat stress (CSDS). Consistent with the neuropharmacological results, overexpression of GHSR in the LS reversed CSDS-induced depressive-like behaviors. Our findings clarify a key role for ghrelin/GHSR signaling in the regulation of chronic stress-induced depressive-like behaviors, which could provide new strategies for the treatment of depression.

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.

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

OBJECTIVE: Obesity-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). METHODS: LysM-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. RESULTS: We 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. CONCLUSIONS: These 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.

Elevated Ghrelin Promotes Hippocampal Ghrelin Receptor Defects in Humanized Amyloid-ß Knockin Mice During Aging.

BACKGROUND: Emerging evidence has revealed that dysregulation of the hormone ghrelin and its receptor, growth hormone secretagogue receptor (GHSR), contributes to the pathogenesis of Alzheimer's disease (AD). Specifically, defective GHSR function and resultant hippocampal ghrelin resistance are linked to hippocampal synaptic injury in AD paradigms. Also, AD patients exhibit elevated ghrelin activation. However, the detailed molecular mechanisms of hippocampal GHSR dysfunction and the relevance of ghrelin elevation to hippocampal ghrelin resistance in AD-relevant pathological settings are not fully understood. OBJECTIVE: In the current study, we employed a recently established mouse line of AD risk [humanized amyloid beta knockin (hAß KI mice), also referred to as a mouse model of late-onset AD in previous literature] to further define the role of ghrelin system dysregulation in the development of AD. METHODS: We employed multidisciplinary techniques to determine the change of plasma ghrelin and the functional status of GHSR in hAß KI mice as well as primary neuron cultures. RESULTS: We observed concurrent plasma ghrelin elevation and hippocampal GHSR desensitization with disease progression. Further examination excluded the possibility that ghrelin elevation is a compensatory change in response to GHSR dysfunction. In contrast, further in vitro and in vivo results show that agonist-mediated overstimulation potentiates GHSR desensitization through enhanced GHSR internalization. CONCLUSIONS: These findings suggest that circulating ghrelin elevation is a pathological event underlying hippocampal GHSR dysfunction, culminating in hippocampal ghrelin resistance and resultant synaptic injury in late-onset AD-related settings.

Does ghrelin regulate intestinal motility in rabbits? An in vitro study using isolated duodenal strips.

Rabbit duodenum has been used for examining the ability of motilin to cause muscle contraction in vitro. A motilin-related peptide, ghrelin, is known to be involved in the regulation of gastrointestinal (GI) motility in various animals, but its ability to cause rabbit GI contraction have not been well examined. The aim of this study is to clarify the action of rat ghrelin and its interaction with motilin in the rabbit duodenum. The mRNA expression of ghrelin and motilin receptors was also examined using RT-PCR. Rat ghrelin (10-9-10-6 M) did not change the contractile activity of the duodenum measured by the mean muscle tonus and area under the curve of contraction waves. In agreement with this result, the distribution of ghrelin receptor mRNA in the rabbit GI tract varied depending on the GI region from which the samples were taken; the expression level in the duodenum was negligible, but that in the esophagus or stomach was significant. On the other hand, motilin (10-10-10-6 M) caused a concentration-dependent contraction by means of increased mean muscle tonus, and consistently, motilin receptor mRNA was expressed heterogeneously depending on the GI region (esophagus = stomach = colon = rectum < duodenum = jejunum = ileum < cecum). Expression level of motilin receptor was comparable to that of ghrelin receptor in the esophagus and stomach. Pretreatment with ghrelin (10-6 M) prior to motilin did not affect the contractile activity of motilin in the duodenum. In conclusion, ghrelin does not affect muscle contractility or motilin-induced contraction in the rabbit duodenum, which is due to the lack of ghrelin receptors. The present in vitro results suggest that ghrelin might not be a regulator of intestinal motility in rabbits.

Nutrient-Sensing Ghrelin Receptor in Macrophages Modulates Bisphenol A-Induced Intestinal Inflammation in Mice.

Bisphenols are environmental toxins with endocrine disruptor activity, yet bisphenol A (BPA) and its analogs are still widely used in manufacturing plastic products. There is evidence showing that BPA elicits inflammation in humans and animals, but the target cell types of BPA are not well understood. In this study, we sought to determine BPA's direct effect on macrophages and BPA immunotoxicity in mouse intestine. Ghrelin is an important nutrient-sensing hormone, acting through its receptor growth hormone secretagogue receptor (GHSR) to regulate metabolism and inflammation. We found that BPA promotes intestinal inflammation, showing increased infiltrating immune cells in colons and enhanced expression of Ghsr and pro-inflammatory cytokines and chemokines, such as Il6 and Ccl2, in colonic mucosa. Moreover, we found that both long- and short-term BPA exposure elevated pro-inflammatory monocytes and macrophages in mouse peripheral blood mononuclear cells (PBMC) and peritoneal macrophages (PM), respectively. To determine the role of GHSR in BPA-mediated inflammation, we generated Ghsr deletion mutation in murine macrophage RAW264.7 using CRISPR gene editing. In wild-type RAW264.7 cells, the BPA exposure promotes macrophage pro-inflammatory polarization and increases Ghsr and cytokine/chemokine Il6 and Ccl2 expression. Interestingly, Ghsr deletion mutants showed a marked reduction in pro-inflammatory cytokine/chemokine expression in response to BPA, suggesting that GHSR is required for the BPA-induced pro-inflammatory response. Further understanding how nutrient-sensing GHSR signaling regulates BPA intestinal immunotoxicity will help design new strategies to mitigate BPA immunotoxicity and provide policy guidance for BPA biosafety.

Down regulation of the hippocampal ghrelin receptor type-1a during electrical kindling-induced epileptogenesis.

Numerous studies have shown that the ghrelin hormone is involved in epileptic conditions. However, the profile of ghrelin or its functional receptor mRNAs in seizure-susceptible brain areas was not assessed during epileptogenesis. Here, we measured the expression levels of the hippocampal ghrelin or its receptor mRNAs during electrical kindling-induced epileptogenesis. The study was conducted on twenty adult male Wistar rats. One tri-polar and two uni-polar electrodes were stereotaxically implanted in the baso-lateral amygdala or skull surface, respectively. Animals were divided into four groups, consisting of two sham groups (sham1 and sham2), and two other groups, which were partially or fully kindled. After the establishment of partial or full kindling, the hippocampi of the animals and that of the corresponding sham groups were removed. A quantitative real-time PCR technique was used to measure the expression levels of ghrelin or its functional receptor mRNAs. The results indicated that the expression levels of ghrelin did not alter in either of the partially or fully kindled rats compared to the corresponding sham groups. Ghrelin receptor (ghrelinR) down regulated, significantly in the fully-kindled rats, compared to sham2 group. Meanwhile, the mRNA expression levels of ghrelinR did not change in partially-kindled rats compared to sham1 group. The outcomes of the current study highlight the crucial, unknown impact of the hippocampal ghrelinR through the development of electrical kindling epileptogenesis, and points out the importance of ghrelinR as a goal to adjust epileptogenic progression.

Ghrelin receptor signaling in health and disease: a biased view.

As allosteric complexes, G-protein-coupled receptors (GPCRs) respond to extracellular stimuli and pleiotropically couple to intracellular transducers to elicit signaling pathway-dependent effects in a process known as biased signaling or functional selectivity. One such GPCR, the ghrelin receptor (GHSR1a), has a crucial role in restoring and maintaining metabolic homeostasis during disrupted energy balance. Thus, pharmacological modulation of GHSR1a bias could offer a promising strategy to treat several metabolism-based disorders. Here, we summarize current evidence supporting GHSR1a functional selectivity in vivo and highlight recent structural data. We propose that precise determinations of GHSR1a molecular pharmacology and pathway-specific physiological effects will enable discovery of GHSR1a drugs with tailored signaling profiles, thereby providing safer and more effective treatments for metabolic diseases.

GHSR1a deficiency suppresses inhibitory drive on dCA1 pyramidal neurons and contributes to memory reinforcement.

Growth hormone secretagogue receptor 1a (GHSR1a)-the receptor for orexigenic hormone ghrelin-is a G protein-coupled receptor that is widely distributed in the brain, including the hippocampus. Studies have demonstrated that genetic deletion of GHSR1a affects memory, suggesting the importance of ghrelin/GHSR1a signaling in cognitive control. However, current reports are controversial, and the mechanism underlying GHSR1a modulation of memory is uncertain. Here, we first report that global GHSR1a knockout enhances hippocampus-dependent memory, facilitates initial LTP in dorsal hippocampal Schaffer Collateral-CA1 synapses, and downregulates Akt activity in the hippocampus. Moreover, we show that the intrinsic excitability of GAD67+ interneurons-rather than neighboring pyramidal neurons in the dCA1-is suppressed by GHSR1a deletion, an effect that is antagonized by acute application of the Akt activator SC79. In addition, the inhibitory postsynaptic currents (IPSCs) on dCA1 pyramidal neurons are selectively reduced in mice with a GHSR1a deficiency. Finally, we demonstrate that selectively increasing the excitability of parvalbumin-expressing interneurons by hM3Dq-DREADDs increases IPSCs on dCA1 pyramidal neurons and normalizes memory in Ghsr1a KO mice. Our findings thus reveal a novel mechanism underlying memory enhancement of GHSR1a deficiency and herein support an adverse effect of GHSR1a signaling in hippocampus-dependent memory processes.

Expressions of ghrelin and GHSR-1a in the corpus luteum and the stimulatory effect of ghrelin on luteal function of pregnant sows.

Studies have shown that ghrelin played direct actions in ovarian function, but the direct role of ghrelin in corpus luteum (CL) of pregnant sows has remained obscure. The study aimed to examine the expressions of ghrelin and its functional receptor (GHSR-1a) in the CL of sows during pregnancy, and evaluate the role of ghrelin in CL function of pregnant sows. Immunohistochemistry analysis showed that ghrelin and GHSR-1a are both predominantly localized in the luteal cells of pregnant sows CL. Strong immunoreactivity for ghrelin and GHSR-1a is detected at days 20 (early) and 50 (middle), but weak immunoreactivity is observed at days 90 (late) post mating. Similarly, there is a significant effect of pregnant phase on the expression (mRNA and protein) of ghrelin and GHSR-1a in the CL, with higher levels at days 20 (early) and 50 (middle), and lower values at 90 (late) post mating. In vitro, treatments of luteal cells with ghrelin (from 0.01 to 10 ng/mL) are promoted cell viability and P4 secretion in a dose-dependent manner. Ghrelin is also accelerated the LH-induced P4 secretion in luteal cells. Moreover, ghrelin is induced the release and mRNA expression of LH, and increased the release of prostaglandin (PG)E2, but reduced the secretion of PGF2α in luteal cells. In conclusion, the presences of ghrelin and GHSR-1a in the porcine CL during pregnancy, and the stimulatory effect of ghrelin on luteal cells suggest positive regulation by ghrelin in CL function of pregnant sows.

Establishment of a Cell Line Stably Expressing the Growth Hormone Secretagogue Receptor to Identify Crocin as a Ghrelin Agonist.

The growth hormone secretagogue receptor-1a (GHSR1a) is the endogenous receptor for ghrelin. Activation of GHSR1a participates in many physiological processes including energy homeostasis and eating behavior. Due to its transitory half-life, the efficacy of ghrelin treatment in patients is restricted; hence the development of new adjuvant therapy is an urgent need. This study aimed to establish a cell line stably expressing GHSR1a, which could be employed to screen potential ghrelin agonists from natural compounds. First, by means of lentiviral transduction, the genome of a human HEK293T cell was modified, and a cell platform stably overexpressing GHSR1a was successfully established. In this platform, GHSR1a was expressed as a fusion protein tagged with mCherry, which allowed the monitoring of the dynamic cellular distribution of GHSR1a by fluorescent microscopy. Subsequently, the authenticity of the GHSR1a mediated signaling was further characterized by using ghrelin and teaghrelin, two molecules known to stimulate GHSR1a. The results indicated that both ghrelin and teaghrelin readily activated GHSR1a mediated signaling pathways, presumably via increasing phosphorylation levels of ERK. The specific GHSR1a signaling was further validated by using SP-analog, an antagonist of GHSR1a as well as using a cell model with the knockdown expression of GHSR1a. Molecular modeling predicted that crocin might be a potential ghrelin agonist, and this prediction was further confirmed by the established platform.

Involvement of POMC neurons in LEAP2 regulation of food intake and body weight.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a newly discovered antagonist of the growth hormone secretagogue receptor (GHSR) and is considered the first endogenous peptide that can antagonize the metabolic actions of ghrelin. The effects of ghrelin administration on feeding behavior, body weight, and energy metabolism involve the activation of orexigenic neurons in the arcuate nucleus (ARC) of the hypothalamus. It is unclear, however, if LEAP2 applied directly to the ARC of the hypothalamus affects these metabolic processes. Here, we show that overexpression of LEAP2 in the ARC through adeno-associated virus (AAV) reduced food intake and body weight in wild-type (WT) mice fed chow and a high-fat diet (HFD) and improved metabolic disorders. LEAP2 overexpression in the ARC overrides both central and peripheral ghrelin action on a chow diet. Interestingly, this AAV-LEAP2 treatment increased proopiomelanocortin (POMC) expression while agouti-related peptide (AGRP)/neuropeptide Y (NPY) and GHSR levels remained unchanged in the hypothalamus. Additionally, intracerebroventricular (i.c.v.) administration of LEAP2 decreased food intake, increased POMC neuronal activity, and repeated LEAP2 administration to mice induced body weight loss. Using chemogenetic manipulations, we found that inhibition of POMC neurons abolished the anorexigenic effect of LEAP2. These results demonstrate that central delivery of LEAP2 leads to appetite-suppressing and body weight reduction, which might require activation of POMC neurons in the ARC.

Deletion of Growth Hormone Secretagogue Receptor in Kisspeptin Neurons in Female Mice Blocks Diet-Induced Obesity.

The gut peptide, ghrelin, mediates energy homeostasis and reproduction by acting through its receptor, growth hormone secretagogue receptor (GHSR), expressed in hypothalamic neurons in the arcuate (ARC). We have shown 17ß-estradiol (E2) increases Ghsr expression in Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons, enhancing sensitivity to ghrelin. We hypothesized that E2-induced Ghsr expression augments KNDy sensitivity in a fasting state by elevating ghrelin to disrupt energy expenditure in females. We produced a Kiss1-GHSR knockout to determine the role of GHSR in ARC KNDy neurons. We found that changes in ARC gene expression with estradiol benzoate (EB) treatment were abrogated by the deletion of GHSR and ghrelin abolished these differences. We also observed changes in metabolism and fasting glucose levels. Additionally, knockouts were resistant to body weight gain on a high fat diet (HFD). Behaviorally, we found that knockouts on HFD exhibited reduced anxiety-like behavior. Furthermore, knockouts did not refeed to the same extent as controls after a 24 h fast. Finally, in response to cold stress, knockout females had elevated metabolic parameters compared to controls. These data indicate GHSR in Kiss1 neurons modulate ARC gene expression, metabolism, glucose homeostasis, behavior, and thermoregulation, illustrating a novel mechanism for E2 and ghrelin to control Kiss1 neurons.

Ablation of Ghrelin Receptor Mitigates the Metabolic Decline of Aging Skeletal Muscle.

The orexigenic hormone ghrelin has multifaceted roles in health and disease. We have reported that ablation of the ghrelin receptor, growth hormone secretagogue receptor (GHS-R), protects against metabolic dysfunction of adipose tissues in aging. Our further observation interestingly revealed that GHS-R deficiency phenocopies the effects of myokine irisin. In this study, we aim to determine whether GHS-R affects the metabolic functions of aging skeletal muscle and whether GHS-R regulates the muscular functions via irisin. We first studied the expression of metabolic signature genes in gastrocnemius muscle of young, middle-aged and old mice. Then, old GHS-R knockout (Ghsr-/-) mice and their wild type counterparts were used to assess the impact of GHS-R ablation on the metabolic characteristics of gastrocnemius and soleus muscle. There was an increase of GHS-R expression in skeletal muscle during aging, inversely correlated with the decline of metabolic functions. Remarkedly the muscle of old GHS-R knockout (Ghsr-/-) mice exhibited a youthful metabolic profile and better maintenance of oxidative type 2 muscle fibers. Furthermore, old Ghsr-/- mice showed improved treadmill performance, supporting better functionality. Also intriguing to note was the fact that old GHS-R-ablated mice showed increased expression of the irisin precursor FNDC5 in the muscle and elevated plasma irisin levels in circulation, which supports a potential interrelationship between GHS-R and irisin. Overall, our work suggests that GHS-R has deleterious effects on the metabolism of aging muscle, which may be at least partially mediated by myokine irisin.

Ghrelin and ghrelin receptor (GHSR) in Chinese alligator, alligator sinensis: Molecular characterization, tissue distribution and mRNA expression changes during the active and hibernating periods.

The Chinese alligator (Alligator sinensis) is a freshwater crocodilian endemic to China. So far, the endocrine regulation of feeding and growth in Chinese alligator is poorly understood. In this study, the molecular structure and tissue expression profiles of ghrelin and its receptor GHSR in the Chinese alligator were characterized for the first time. The full-length cDNA of ghrelin was 1770 bp, including a 37 bp 5 '-UTR (untranslated region), a 435 bp ORF (open reading frame) and a 1298 bp 3 '-UTR. The ORF encodes a ghrelin precursor, which consists of 145 amino acid residues, including a signal peptide with 52 amino acid residues at the N-terminus, a mature peptide with 28 amino acid residues, and a possibly obestain at the C-terminus. The full-length cDNA of GHSR was 3961 bp, including a 5'-UTR of 375-bp, an ORF of 1059-bp and a 3' -UTR of 2527-bp. The ORF encodes a protein of 352 amino acid residues containing seven transmembrane domains, with multiple N glycosylation modification sites and conserved cysteine residue sites. The active core "GSSF" of Chinese alligator ghrelin was identical to that of mammals and birds, and the ghrelin binding site of GHSR was similar to that of mammals. The amino acid sequences of both ghrelin and GHSR share high identity with American alligator (Alligator mississippiensis) and birds. Ghrelin was highly expressed in cerebrum, mesencephalon, hypothalamus and multiple peripheral tissues, including lung, stomach and intestine, suggesting that it could play functions in paracrine and/or autocrine manners in addition to endocrine manner. GHSR expression level was higher in hypothalamus, epencephalon and medulla oblongata, and moderate in multiple peripheral tissues including lung, kindey, stomach and oviduct, implicating that ghrelin/GHSR system may participate in the regulation of energy balance, food intake, water and mineral balance, gastrointestinal motility, gastric acid secretion and reproduction. During hibernation, the expression of ghrelin and GHSR in the brain was significantly increased, while ghrelin was significantly decreased in heart, liver, lung, stomach, pancreas and ovary, and GHSR was significantly decreased in heart, liver, spleen, lung, kindey, stomach, ovary and oviduct. These temporal changes in ghrelin and GHSR expression could facilitate the physiological adaption to the hibernation of Chinese alligator. Our study could provide basic data for further studies on the regulation of feeding, physiological metabolism and reproduction of Chinese alligator, which could also be useful for the improvement of artificial breeding of this endangered species.