Discovery of a dual-action small molecule that improves neuropathological features of Alzheimer's disease mice.

Alzheimer's disease (AD) is characterized by complex, multifactorial neuropathology, suggesting that small molecules targeting multiple neuropathological factors are likely required to successfully impact clinical progression. Acid sphingomyelinase (ASM) activation has been recognized as an important contributor to these neuropathological features in AD, leading to the concept of using ASM inhibitors for the treatment of this disorder. Here we report the identification of KARI 201, a direct ASM inhibitor evaluated for AD treatment. KARI 201 exhibits highly selective inhibition effects on ASM, with excellent pharmacokinetic properties, especially with regard to brain distribution. Unexpectedly, we found another role of KARI 201 as a ghrelin receptor agonist, which also has therapeutic potential for AD treatment. This dual role of KARI 201 in neurons efficiently rescued neuropathological features in AD mice, including amyloid beta deposition, autophagy dysfunction, neuroinflammation, synaptic loss, and decreased hippocampal neurogenesis and synaptic plasticity, leading to an improvement in memory function. Our data highlight the possibility of potential clinical application of KARI 201 as an innovative and multifaceted drug for AD treatment.

Ghrelin/GHSR System in Depressive Disorder: Pathologic Roles and Therapeutic Implications.

Depression is the most common chronic mental illness and is characterized by low mood, insomnia, and affective disorders. However, its pathologic mechanisms remain unclear. Numerous studies have suggested that the ghrelin/GHSR system may be involved in the pathophysiologic process of depression. Ghrelin plays a dual role in experimental animals, increasing depressed behavior and decreasing anxiety. By combining several neuropeptides and traditional neurotransmitter systems to construct neural networks, this hormone modifies signals connected to depression. The present review focuses on the role of ghrelin in neuritogenesis, astrocyte protection, inflammatory factor production, and endocrine disruption in depression. Furthermore, ghrelin/GHSR can activate multiple signaling pathways, including cAMP/CREB/BDNF, PI3K/Akt, Jak2/STAT3, and p38-MAPK, to produce antidepressant effects, given which it is expected to become a potential therapeutic target for the treatment of depression.

GHS-R1a deficiency protects against lipopolysaccharide-induced spatial memory impairment in mice.

Neuroinflammation has been implicated in cognitive deficits of neurological and neurodegenerative diseases. There is abundant evidence that the application of ghrelin, an orexigenic hormone regulating appetite and energy balance, abrogates neuroinflammation and rescues associated memory impairment. However, the underlying mechanism is uncertain. In this study, we find that both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of lipopolysaccharide (LPS) impairs spatial memory in mice. LPS treatment causes neuroinflammation and microglial activation in the hippocampus. Ghsr1a deletion suppresses LPS-induced microglial activation and neuroinflammation, and rescued LPS-induced memory impairment. Our findings thus suggest that GHS-R1a signaling may promote microglial immunoactivation and contribute to LPS-induced neuroinflammation. GHS-R1a may be a new therapeutic target for cognitive dysfunction associated with inflammatory conditions.

Loss of LEAP-2 alleviates obesity-induced myocardial injury by regulating macrophage polarization.

BACKGROUND: Obesity is a serious public health issue worldwide, which is a risk factor of cardiovascular disorders. Obesity has been shown to be associated with subclinical myocardial injury, increasing the risk of heart failure. Our study aims to explore novel mechanisms underlying obesity-induced myocardial injury. METHODS: Mice were fed a high-fat diet (HFD) to establish a mouse model of obesity, and serum levels of TG, TCH, LDL, CK-MB, LDH, cTnI and BNP were examined. Inflammatory response was evaluated by determining the expression and secretion of proinflammatory cytokines IL-1ß and TNF-α. Macrophage infiltration in the heart was examined by IHC staining, and H&E staining was applied to evaluate myocardial injury. Primary peritoneal macrophages were isolated from mice and treated with palmitic acid (PA). Macrophage polarization was evaluated by determine the expression of CCL2, iNOS, CD206 and arginase I via Western blot, RT-qPCR, and flow cytometry. Co-IP assays were performed to examine the interaction between LEAP-2, GHSR and ghrelin. RESULTS: Hyperlipidemia, increased proinflammatory cytokines and myocardial injury were observed in mice with obesity, and silencing of LEAP-2 ameliorated HFD-induced hyperlipidemia, inflammation, and myocardial injury. Moreover, HFD-induced macrophage infiltration and M1 polarization were reversed by LEAP-2 knockdown in mice. Furthermore, silencing of LEAP-2 suppressed PA-induced M1 polarization but enhanced M2 polarization in vitro. LEAP-2 interacted with GHSR in macrophages, and knockdown of LEAP-2 promoted the interaction of GHSR and ghrelin. Overexpression of ghrelin enhanced LEAP-1 silencing-mediated suppression of inflammatory response and upregulation of M2 polarization in PA-induced macrophages. CONCLUSION: Knockdown of LEAP-2 ameliorates obesity-induced myocardial injury via promoting M2 polarization.

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.

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.

The ghrelin receptor GHSR has two efficient agonists in the lobe-finned fish Latimeria chalumnae.

The peptide hormone ghrelin (an agonist) and LEAP2 (an antagonist) play important functions in energy metabolism via their receptor GHSR, an A-class G protein-coupled receptor. Ghrelin, LEAP2, and GHSR are widely present from fishes to mammals. However, our recent study suggested that fish GHSRs have different binding properties to ghrelin: a GHSR from the lobe-finned fish Latimeria chalumnae (coelacanth) is efficiently activated by ghrelin, but GHSRs from the ray-finned fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost binding to ghrelin. Do fish GHSRs use another peptide as their agonist? In the present study we tested to two fish motilins from D. rerio and L. chalumnae because motilin is distantly related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the fish GHSR from L. chalumnae bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in the ray-finned fish D. rerio and L. crocea, but is an efficient agonist for GHSR in the lobe-finned fish L. chalumnae, one of the closest fish relatives of tetrapods. The results of present study suggested that GHSR might have two efficient agonists, ghrelin and motilin, in ancient fishes; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins.

Potential role of ghrelin in the regulation of inflammation.

Several diseases are caused or progress due to inflammation. In the past few years, accumulating evidence suggests that ghrelin, a gastric hormone of 28-amino acid residue length, exerts protective effects against inflammation by modulating the related pathways. This review focuses on ghrelin's anti-inflammatory and potential therapeutic effects in neurological, cardiovascular, respiratory, hepatic, gastrointestinal, and kidney disorders. Ghrelin significantly alleviates excessive inflammation and reduces damage to different target organs mainly by reducing the secretion of inflammatory cytokines, including interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α), and inhibiting the nuclear factor kappa-B (NF-κB) and NLRP3 inflammasome signaling pathways. Ghrelin also regulates inflammation and apoptosis through the p38 MAPK/c-Jun N-terminal kinase (JNK) signaling pathway; restores cerebral microvascular integrity, and attenuates vascular leakage. Ghrelin activates the phosphoInositide-3 kinase (PI3K)/protein kinase B (Akt) pathway and inhibits inflammatory responses in cardiovascular diseases and acute kidney injury. Some studies show that ghrelin exacerbates colonic and intestinal manifestations of colitis. Interestingly, some inflammatory states, such as non-alcoholic steatohepatitis, inflammatory bowel diseases, and chronic kidney disease, are often associated with high ghrelin levels. Thus, ghrelin may be a potential new therapeutic target for inflammation-related diseases.

Metabolic, neuroendocrine and behavioral effects of social defeat in male and female mice using the chronic non-discriminatory social defeat stress model.

Stress-related disorders predominately affect females, yet preclinical models of chronic stress exclusively use males especially in models where social stressors are studied. Here, we implemented a 21-day novel social defeat paradigm in which a female and male C57 intruder are simultaneously placed in the cage of a territorial, resident CD-1 male mouse, and the resident proceeds to attack both intruders. Mice were given access to a regular laboratory diet, high in carbohydrates, and a palatable diet, high in fat. Chronic social defeat stress using this paradigm resulted in increased caloric intake in male and female mice, with the effects being more pronounced in females. We observed sex differences in high fat diet intake in response to stress, which was correlated with higher levels of plasma ghrelin observed in female mice but not male mice. Furthermore, females exposed to chronic stress displayed changes in growth hormone secretatogue receptor (ghsr) and neuropeptide-y (npy) expression in the arcuate nucleus of the hypothalamus, potentially increasing ghrelin sensitivity and inducing changes in diet choice and caloric intake. Behavioral results show that females tended to spend more time interacting during the social interaction test, compared to males who displayed higher vigilance towards the stranger mouse. Overall, our results highlight unique neurometabolic alterations in female mice in response to stress that is not present in male mice and may be important for coping with chronic stress and sustaining reproductive function.

Systemic Ghrelin Treatment Induces Rapid, Transient, and Asymmetric Changes in the Metabolic Activity of the Mouse Brain.

INTRODUCTION: Ghrelin regulates a variety of functions by acting in the brain. The targets of ghrelin in the mouse brain have been mainly mapped using immunolabeling against c-Fos, a transcription factor used as a marker of cellular activation, but such analysis has several limitations. Here, we used positron emission tomography in mice to investigate the brain areas responsive to ghrelin. METHODS: We analyzed in male mice the brain areas responsive to systemically injected ghrelin using positron emission tomography imaging of 18F-fluoro-2-deoxyglucose (18F-FDG) uptake, an indicator of metabolic rate. Additionally, we studied if systemic administration of fluorescent ghrelin or native ghrelin displays symmetric accessibility or induction of c-Fos, respectively, in the brain of male mice. RESULTS: Ghrelin increased 18F-FDG uptake in few specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain at 0-10-min posttreatment. At the 10-20 and 20-30 min posttreatment, ghrelin induced mixed changes in 18F-FDG uptake in specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain, as well as in areas of the olfactory areas, hippocampal and retrohippocampal regions, hypothalamus, pons, medulla, and even the cerebellum. Ghrelin-induced changes in 18F-FDG uptake were transient and asymmetric. Systemically administrated fluorescent-ghrelin-labeled midline brain areas known to contain fenestrated capillaries and the hypothalamic arcuate nucleus, where a symmetric labeling was observed. Ghrelin treatment also induced a symmetric increased c-Fos labeling in the arcuate nucleus. DISCUSSION/CONCLUSION: Systemically injected ghrelin transiently and asymmetrically affects the metabolic activity of the brain of male mice in a wide range of areas, in a food intake-independent manner. The neurobiological bases of such asymmetry seem to be independent of the accessibility of ghrelin into the brain.

Molecular cloning and analysis of the ghrelin/GHSR system in Xenopus tropicalis.

Ghrelin is a gut-derived peptide with several physiological functions, including feeding, gastrointestinal motility, and hormonal secretion. Recently, a host defense peptide, liver-expressed antimicrobial peptide-2 (LEAP2), was reported as an endogenous antagonist of growth hormone secretagogue receptor (GHS-R). The physiological relevance of the molecular LEAP2-GHS-R interaction in mammals has been explored; however, studies on non-mammals are limited. Here, we report the identification and functional characterization of ghrelin and its related molecules in Western clawed frog (Xenopus tropicalis), a known model organism. We first identified cDNA encoding X. tropicalis ghrelin and GHS-R. RT-qPCR revealed that ghrelin mRNA expression was most abundant in the stomach. GHS-R mRNA was widely distributed in the brain and peripheral tissues, and a relatively strong signal was observed in the stomach and intestine. In addition, LEAP2 was mainly expressed in intestinal tissues at higher levels than in the liver. In functional analysis, X. tropicalis ghrelin and human ghrelin induced intracellular Ca2+ mobilization with EC50 values in the low nanomolar range in CHO-K1 cells expressing X. tropicalis GHS-R. Furthermore, ghrelin-induced GHS-R activation was antagonized with IC50 values in the nanomolar range by heterologous human LEAP2. We also validated the expression of ghrelin and feeding-related factors under fasting conditions. After 2 days of fasting, no changes in ghrelin mRNA levels were observed in the stomach, but GHS-R mRNA levels were significantly increased, associated with significant downregulation of nucb2. In addition, LEAP2 upregulation was observed in the duodenum. These results provide the first evidence that LEAP2 functions as an antagonist of GHS-R in the anuran amphibian X. tropicalis. It has also been suggested that the ghrelin/GHS-R/LEAP2 system may be involved in energy homeostasis in X. tropicalis.

GHSR controls food deprivation-induced activation of CRF neurons of the hypothalamic paraventricular nucleus in a LEAP2-dependent manner.

OBJECTIVE: Prolonged fasting is a major challenge for living organisms. An appropriate metabolic response to food deprivation requires the activation of the corticotropin-releasing factor-producing neurons of the hypothalamic paraventricular nucleus (PVHCRF neurons), which are a part of the hypothalamic-pituitary-adrenal axis (HPA), as well as the growth hormone secretagogue receptor (GHSR) signaling, whose activity is up- or down-regulated, respectively, by the hormones ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2). Since ghrelin treatment potently up-regulates the HPA axis, we studied the role of GHSR in mediating food deprivation-induced activation of the PVHCRF neurons in mice. METHODS: We estimated the activation of the PVHCRF neurons, using immuno-staining against CRF and the marker of neuronal activation c-Fos in brain sections, and assessed plasma levels of corticosterone and glucose in different pharmacologically or genetically manipulated mouse models exposed, or not, to a 2-day food deprivation protocol. In particular, we investigated ad libitum fed or food-deprived male mice that: (1) lacked GHSR gene expression, (2) had genetic deletion of the ghrelin gene, (3) displayed neurotoxic ablation of the hypothalamic arcuate nucleus, (4) were centrally treated with an anti-ghrelin antibody to block central ghrelin action, (5) were centrally treated with a GHSR ligand that blocks ghrelin-evoked and constitutive GHSR activities, or (6) received a continuous systemic infusion of LEAP2(1-12). RESULTS: We found that food deprivation results in the activation of the PVHCRF neurons and in a rise of the ghrelin/LEAP2 molar ratio. Food deprivation-induced activation of PVHCRF neurons required the presence and the signaling of GHSR at hypothalamic level, but not of ghrelin. Finally, we found that preventing the food deprivation-induced fall of LEAP2 reverses the activation of the PVHCRF neurons in food-deprived mice, although it has no effect on body weight or blood glucose. CONCLUSION: Food deprivation-induced activation of the PVHCRF neurons involves ghrelin-independent actions of GHSR at hypothalamic level and requires a decrease of plasma LEAP2 levels. We propose that the up-regulation of the actions of GHSR associated to the fall of plasma LEAP2 level are physiologically relevant neuroendocrine signals during a prolonged fasting.

Ghrelin rapidly elevates protein synthesis in vitro by employing the rpS6K-eEF2K-eEF2 signalling axis.

Activated ghrelin receptor GHS-R1α triggers cell signalling pathways that modulate energy homeostasis and biosynthetic processes. However, the effects of ghrelin on mRNA translation are unknown. Using various reporter assays, here we demonstrate a rapid elevation of protein synthesis in cells within 15-30 min upon stimulation of GHS-R1α by ghrelin. We further show that ghrelin-induced activation of translation is mediated, at least in part, through the de-phosphorylation (de-suppression) of elongation factor 2 (eEF2). The levels of eEF2 phosphorylation at Thr56 decrease due to the reduced activity of eEF2 kinase, which is inhibited via Ser366 phosphorylation by rpS6 kinases. Being stress-susceptible, the ghrelin-mediated decrease in eEF2 phosphorylation can be abolished by glucose deprivation and mitochondrial uncoupling. We believe that the observed burst of translation benefits rapid restocking of neuropeptides, which are released upon GHS-R1α activation, and represents the most time- and energy-efficient way of prompt recharging the orexigenic neuronal circuitry.

Ghrelin improves cognition via activation of the cAMP- CREB signalling pathway in depressed male C57BL/6J mice.

BACKGROUND: Depression leads to a cognitive decline and decreases in ghrelin are observed in depression. Ghrelin affects the level of Brain-derived nerve growth factor (BDNF) through the cAMP-CREB signalling pathway, and lower BDNF levels lead to cognitive decline. Therefore, it is reasonable to assume that in depression, lower ghrelin causes a decrease in BDNF levels and cognitive decline though the cAMP- CREB signalling pathway. METHODS: A total of 120 C57BL/6J male mice were randomly divided into six groups of 20 mice: non-depression groups (sham group, ghrelin group, and ghrelin + (D-lys3)-GHRP-6 group) and depression groups (depression group, depression + ghrelin group and depression + ghrelin + (D-lys3)-GHRP group). A depression mouse model was established by injecting normal saline, ghrelin or ghrelin + (D-lys3) -GHRP-6 into the lateral ventricle of each group. Cognition, hippocampal long-term potentiation (LTP), ghrelin mRNA and protein level, BDNF level and CREB level in the hippocampus were detected. RESULTS: In the depression mouse model groups, all comparison indexes (cognition and hippocampal levels of LTP, ghrelin mRNA and proteins, and BDNF and CREB) had significant negative changes. In the mice with depression, ghrelin or ghrelin + (D-lys3)-GHRP-6 was injected, and all the comparison indicators showed significant positive changes. Supplementation of ghrelin+(D-lys3))-GHRP-6 resulted in more significant positive changes in all comparison indexes than those of ghrelin alone. CONCLUSIONS: In the depression model, lower ghrelin causes hippocampal BDNF to decrease and results in cognitive decline via the cAMP-CREB signalling pathway.

Ligand-Free Signaling of G-Protein-Coupled Receptors: Physiology, Pharmacology, and Genetics.

G-protein-coupled receptors (GPCRs) are ubiquitous sensors and regulators of cellular functions. Each GPCR exists in complex aggregates with multiple resting and active conformations. Designed to detect weak stimuli, GPCRs can also activate spontaneously, resulting in basal ligand-free signaling. Agonists trigger a cascade of events leading to an activated agonist-receptor G-protein complex with high agonist affinity. However, the ensuing signaling process can further remodel the receptor complex to reduce agonist affinity, causing rapid ligand dissociation. The acutely activated ligand-free receptor can continue signaling, as proposed for rhodopsin and µ opioid receptors, resulting in robust receptor activation at low agonist occupancy with enhanced agonist potency. Continued receptor stimulation can further modify the receptor complex, regulating sustained ligand-free signaling-proposed to play a role in opioid dependence. Basal, acutely agonist-triggered, and sustained elevated ligand-free signaling could each have distinct functions, reflecting multi-state conformations of GPCRs. This review addresses basal and stimulus-activated ligand-free signaling, its regulation, genetic factors, and pharmacological implications, focusing on opioid and serotonin receptors, and the growth hormone secretagogue receptor (GHSR). The hypothesis is proposed that ligand-free signaling of 5-HT2A receptors mediate therapeutic effects of psychedelic drugs. Research avenues are suggested to close the gaps in our knowledge of ligand-free GPCR signaling.

Virus-mediated GHS-R1a expression in the basolateral amygdala blocks extinction of conditioned taste aversion memory in rats.

Ghrelin is an orexigenic gastric hormone that promotes feeding behaviors and regulating energy homeostasis in both humans and rodents. Our previous studies have shown that ghrelin, when locally infused into the basolateral amygdala (BLA), blocks both acquisition and extinction of conditioned taste aversion (CTA) memory in rats. In this study, we further investigated the effect of virus-mediated overexpression of ghrelin receptor growth hormone secretagogue receptor 1a (GHS-R1a) in BLA pyramidal neurons on CTA memory processes. We found that upregulation of GHS-R1a expression in BLA pyramidal neurons repressed CTA extinction while it had no effect on CTA acquisition. In addition, we reported that local infusion of the endogenous GHS-R1a antagonist, liver-expressed antimicrobial peptide 2 (LEAP2), in the BLA abolished the inhibitory effect of increased GHS-R1a on CTA memory extinction. Those findings provide new supportive evidence that ghrelin/GHS-R1a signaling in the BLA circuit shapes emotional memory processes.

GHS-R1a activity suppresses synaptic function of primary cultured hippocampal neurons.

Ghrelin is an orexigenic gastric hormone that promotes feeding behaviors and regulates energy homeostasis in both humans and rodents. Studies have reported intriguing yet conflicting roles that ghrelin and its receptor growth hormone secretagogue receptor 1a (GHS-R1a) play in regulating multiple brain functions, such as learning and memory; however, the underlying mechanism is largely unknown. In this study, we investigated the effect of ghrelin incubation and virus-mediated GHS-R1a overexpression on synaptic functions of primary cultured hippocampal neurons. Our results demonstrated that ghrelin pre-treatment for 24 h, with a concentration of either 4 nM or 200 nM, suppressed the frequency of miniature excitatory postsynaptic currents (mEPSCs), the frequency and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Similarly, GHS-R1a overexpression inhibited both the frequency and the amplitude of mEPSCs, and mIPSCs frequency. Moreover, our in vitro Ca2+-image study with Rhod-3AM reveals that ghrelin pre-treatment for either 3 h or 24 h suppressed glutamate-induced elevation of cytoplasmic [Ca2+]. Our findings thus suggest that GHS-R1a signaling inhibits synaptic function of hippocampal neurons, which may contribute to the blocking effect of ghrelin on memory formation.

Fluorescent P-Hydroxyphosphole for Peptide Labeling through P-N Bond Formation.

Synthesis of fluorescent P-hydroxybinaphtylphosphole-oxide or -sulfide was achieved by trapping a binaphtyl dianion with methyl dichlorophosphite or P-(N,N-diethylamino)dichlorophosphine, followed by oxidation or sulfuration of the P-center. After saponification or acid hydrolysis, the P-hydroxyphospholes were coupled to peptides using the coupling agent BOP, under the conditions required for the synthesis in solution or on a solid support. This new method was illustrated by the labeling of the JMV2959, a potent antagonist of the Growth Hormone Secretagogue Receptor type 1a (GHS-R1a). The labeled conjugates were used to characterize GHSR ligands by competition assays, based on Fluorescence Resonance Energy Transfer (FRET). Such P-hydroxyphosphole-oxide or -sulfide constitute a promising new class of compact fluorophores with large Stokes shift, for labeling biomolecules by grafting through the phosphorus atom.

Stimulation of endogenous pulsatile growth hormone secretion by activation of growth hormone secretagogue receptor reduces the fat accumulation and improves the insulin sensitivity in obese mice.

Obese individuals often show low growth hormone (GH) secretion, which leads to reduced lipid mobilization and further fat accumulation. Pharmacological approaches to increase GH levels in obese individuals by GH injection or GH-releasing hormone receptor agonist showed promising effects on fat reduction. However, side effects on glucose metabolism and the heavy costs on making large peptides hindered their clinical application. Here, we tested whether stimulation of endogenous GH secretion by a synthetic GH secretagogue receptor (GHSR) agonist, hexarelin, improved the metabolism in a hyperphagic obese mouse model. Male melanocortin 4 receptor knockout mice (MC4RKO) were pair-fed and received continuous hexarelin (10.56 µg/day) or vehicle infusion by an osmotic pump for 3-4 weeks. Hexarelin treatment significantly increased the pulsatile GH secretion without detectable alteration on basal GH secretion in MC4RKO mice. The treated mice showed increased lipolysis and lipid oxidation in the adipose tissue, and reduced de novo lipogenesis in the liver, leading to reduced visceral fat mass, reduced triglyceride content in liver, and unchanged circulating free fatty acid levels. Importantly, hexarelin treatment improved the whole-body insulin sensitivity but did not alter glucose tolerance, insulin levels, or insulin-like growth factor 1 (IGF-1) levels. The metabolic effects of hexarelin were likely through the direct action of GH, as indicated by the increased expression level of genes involved in GH signaling pathways in visceral adipose tissues and liver. In conclusion, hexarelin treatment stimulated the pulsatile GH secretion and reduced the fat accumulation in visceral depots and liver in obese MC4RKO mice with improved insulin sensitivity without altered levels of insulin or IGF-1. It provides evidence for managing obesity by enhancing pulsatile GH secretion through activation of GHSR in the pituitary gland.

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.