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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 (PVHWe estimated the activation of the PVHWe found that food deprivation results in the activation of the PVHFood deprivation-induced activation of the PVH

International audience; There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Pavlovian stimuli predictive of appetitive outcomes can exert a powerful influence on the selection and initiation of action, a phenomenon termed outcome-selective Pavlovian-instrumental transfer (sPIT). Rodent studies suggest that sPIT is insensitive to motivational downshift induced by outcome devaluation, an effect that is, however, relatively underexplored.Here we examined in detail the effects of distinct shifts in motivation from hunger to a state of relative satiety on sPIT in rats.A motivational downshift by outcome-specific devaluation immediately prior to testing markedly reduced overall lever responding and magazine entries but left intact the sPIT effect. A motivational downshift prior testing by (1) giving ad libitum rather than restricted access to maintenance diet in the home cage for 24 hours or by (2) a systemic blockade of hormone secretagogue receptor subtype 1A receptors to inhibit orexigenic actions of ghrelin both reduced overall lever responding and magazine entries. Moreover, these latter motivational downshifts reduced the sPIT effect; however, the sizes of the sPIT effects were still large.Collectively, our rodent findings indicate that major effects of various motivational downshifts are overall inhibition of lever pressing and magazine approach, possibly reflecting reduced general motivation. The observed effects of motivational downshifts on sPIT have implications with regard to the role of general motivating effects in sPIT and to the contribution of Pavlovian-instrumental interactions to excessive food seeking as well as obesity in humans.

The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (CaV2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on CaV2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of CaV2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on CaV2.2 currents, and that the GHSRbinding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR., Instituto Multidisciplinario de Biología Celular

International audience; GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4 and 5, 2 this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.

International audience; We present herein the synthesis of biotin-functionalized polymers (BNAPols) that have been developed for the fixation of membrane proteins (MPs) onto surfaces. BNAPols were synthesized by free-radical polymerization of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived amphiphilic monomer in the presence of a thiol-based transfer agent with an azido group. Then a Huisgen-cycloaddition reaction was performed with Biotin-(PEG) 8-alkyne that resulted in formation of the biotinylated polymers. The designed structure of BNAPols was confirmed by NMR spectroscopy, and a HABA/avidin assay was used for estimating the percentage of biotin grafted on the polymer end chain. The colloidal characterization of these biotin-functionalized polymers was done using both dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. These BNAPols were used to stabilize a model G protein-coupled receptor (GPCR), the human Growth Hormone Secretagogue Receptor (GHSR), out of its membrane environment. Subsequent immobilization of the BNAPols:GHSR complex onto a streptavidin-coated surface allowed screening of ligands based both on their ability to bind the immobilized receptor and to trigger GHSR conformational changes using the fluorescence energy transfer (FRET)-based assay. This opens the way to the use of biotinylated NAPols to immobilize functional, unmodified, membrane proteins, providing original sensor devices for multiple applications including innovative ligand screening assays.

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo.

Background Cachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose-limiting side effect of cancer chemotherapy. Skeletal muscle loss is one of the main characteristics of cachexia that significantly contributes to the functional muscle impairment. Calcium-dependent signaling pathways are believed to play an important role in skeletal muscle decline observed in cachexia, but whether intracellular calcium homeostasis is affected in this situation remains uncertain. Growth hormone secretagogues (GHS), a family of synthetic agonists of ghrelin receptor (GHS-R1a), are being developed as a therapeutic option for cancer cachexia syndrome; however, the exact mechanism by which GHS interfere with skeletal muscle is not fully understood. Methods By a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast-twitch extensor digitorum longus (EDL) muscle of adult rats with cisplatin-induced cachexia and established the potential beneficial effects of two GHS (hexarelin and JMV2894) at this level. Additionally, in vivo measures of grip strength and of ultrasonography recordings allowed us to evaluate the functional impact of GHS therapeutic intervention. Results Cisplatin-treated EDL muscle fibres were characterized by a ~18% significant reduction of the muscle weight and fibre diameter together with an up-regulation of atrogin1/Murf-1 genes and a down-regulation of Pgc1-a gene, all indexes of muscle atrophy, and by a two-fold increase in resting intracellular calcium, [Ca2+]i, compared with control rats. Moreover, the amplitude of the calcium transient induced by caffeine or depolarizing high potassium solution as well as the store-operated calcium entry were ~50% significantly reduced in cisplatin-treated rats. Calcium homeostasis dysregulation parallels with changes of functional ex vivo (excitability and resting macroscopic conductance) and in vivo (forelimb force and muscle volume) outcomes in cachectic animals. Administration of hexarelin or JMV2894 markedly reduced the cisplatin-induced alteration of calcium homeostasis by both common as well as drug-specific mechanisms of action. This effect correlated with muscle function preservation as well as amelioration of various atrophic indexes, thus supporting the functional impact of GHS activity on calcium homeostasis. Conclusions Our findings provide a direct evidence that a dysregulation of calcium homeostasis plays a key role in cisplatin-induced model of cachexia gaining insight into the etiopathogenesis of this form of muscle wasting. Furthermore, our demonstration that GHS administration efficaciously prevents cisplatin-induced calcium homeostasis alteration contributes to elucidate the mechanism of action through which GHS could potentially ameliorate chemotherapy-associated cachexia.

Objective: The octanoylated peptide hormone ghrelin regulates appetite and glycaemic control. Des-acyl ghrelin abolishes some effects of ghrelin, but does not bind to ghrelin receptor. LEAP2 is a novel ligand for ghrelin receptor that blocks the effects of ghrelin. Some evidences show that plasma levels of these peptides are altered in adults with obesity, but their levels in childhood obesity remain poorly studied. Therefore, the objective of this study was to assess fasting plasma levels of ghrelin, des-acyl ghrelin and LEAP2 in children with normoweight, overweight/obesity and their association with different anthropometric and metabolic variables. Design: A total of 42 females and 40 males, ages 3–12 years old were enrolled as a cross-sectional cohort. Results: Plasma levels of des-acyl ghrelin and LEAP2 (but not ghrelin) were lower and ghrelin/des-acyl ghrelin ratio was higher in children with overweight/obesity. Des-acyl ghrelin negatively correlated with age, BMI z-score, insulin and HOMA index, and the correlations were stronger in children with overweight/obesity. LEAP2 levels negatively correlated with BMI z-score. No gender differences were found. Conclusions: Our findings suggest that ghrelin tone is increased in childhood obesity, due to a decrease on plasma levels of des-acyl ghrelin and LEAP2, and that des-acyl ghrelin is associated to insulin resistance, particularly in children with overweight/obesity., Facultad de Ciencias Médicas, Instituto Multidisciplinario de Biología Celular, Facultad de Ciencias Exactas

Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.

The ghrelin system has received substantial recognition as a potential target for novel anti-seizure drugs. Ghrelin receptor (ghrelin-R) signaling is complex, involving G&alpha, q/11, G&alpha, i/o, G&alpha, 12/13, and &beta, arrestin pathways. In this study, we aimed to deepen our understanding regarding signaling pathways downstream the ghrelin-R responsible for mediating anticonvulsive effects in a kindling model. Mice were administered the proconvulsive dopamine 1 receptor-agonist, SKF81297, to gradually induce a kindled state. Prior to every SKF81297 injection, mice were treated with a ghrelin-R full agonist (JMV-1843), a G&alpha, q and G&alpha, 12 biased ligand unable to recruit &beta, arrestin (YIL781), a ghrelin-R antagonist (JMV-2959), or saline. Mice treated with JMV-1843 had fewer and less severe seizures compared to saline-treated controls, while mice treated with YIL781 experienced longer and more severe seizures. JMV-2959 treatment did not lead to differences in seizure severity and number. Altogether, these results indicate that the G&alpha, q or G&alpha, 12 signaling pathways are not responsible for mediating JMV-1843&prime, s anticonvulsive effects and suggest a possible involvement of &beta, arrestin signaling in the anticonvulsive effects mediated by ghrelin-R modulation.

Growth hormone deficiency (GHD) is a severe pathology that greatly affects the quality of life, and increases morbidity and mortality of patients owing to the augmentation of cardiovascular events. Treatment of GHD is challenging, mainly because there is no specific characteristic sign or symptom that can be used to make a clear diagnosis. There is need for an unequivocal diagnosis of GHD to avoid unnecessary treatment with GH, because the available provocative tests (GH stimulation tests) are not specific and sensitive enough, and are contraindicated in some patients. Ghrelin is an endogenous peptide that stimulates GH secretion by interacting with a G-protein-coupled receptor named ghrelin receptor (GH secretagogue receptor 1a, GHS-R1a). Given this, a GH stimulation test using ghrelin or its analogues appears to be attractive. In this paper, a modified tripeptide first named JMV-1843 in the laboratory is briefly presented. It is potent and selective in stimulating the release of GH and is orally active. It has been recently commercialised for the diagnosis of adult GH deficiency under the tradename Macrilen. The test using this compound appears to be reliable, well tolerated, and simple.

TLQP-21 is a neuropeptide which has been implicated in regulation of nociception and other relevant physiologic functions. Although recent studies identified C3a and gC1q receptors as targets for TLQP-21, its intracellular molecular mechanisms of action remain largely unidentified. Our aim was (i) to explore the intracellular signaling pathway(s) activated by JMV5656, a novel derivative of TLQP-21, in RAW264.7 macrophages, and (ii) to assess linkages of these pathways with its purported receptors. JMV5656 stimulated, in a dose-dependent fashion, a rapid and transient increase in intracellular Ca2+ concentrations in RAW264.7 cells; repeated exposure to the peptide resulted in a lower response, suggesting a possible desensitization mechanism of the receptor. In particular, JMV5656 increased cytoplasmic Ca2+ levels by a PLC-dependent release of Ca2+ from the endoplasmic reticulum. STIM proteins and Orai Ca2+ channels were activated and played a crucial role. In fact, treatment of the cells with U73122 and thapsigargin modulated the increase of intracellular Ca2+ levels stimulated by JMV5656. Moreover, in RAW264.7 cells intracellular Ca2+ increases did not occur through the binding of JMV5656 to the C3a receptor, since the increase of intracellular Ca2+ levels induced by JMV5656 was not affected by specific siRNA against C3aR. In summary, our study provides new indications for the downstream effects of JMV5656 in macrophages, suggesting that it could activate receptors different from the C3aR.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

International audience

Ghrelin is a potent orexigenic peptide hormone that acts through the growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor highly expressed in the hypothalamus. In vitro studies have shown that GHSR displays a high constitutive activity, whose physiological relevance is uncertain. As GHSR gene expression in the hypothalamus is known to increase in fasting conditions, we tested the hypothesis that constitutive GHSR activity at the hypothalamic level drives the fasting-induced hyperphagia. We found that refed wild-type (WT) mice displayed a robust hyperphagia that continued for 5 days after refeeding and changed their food intake daily pattern. Fasted WT mice showed an increase in plasma ghrelin levels, as well as in GHSR expression levels and ghrelin binding sites in the hypothalamic arcuate nucleus. When fasting-refeeding responses were evaluated in ghrelin- or GHSR-deficient mice, only the latter displayed an ∼15% smaller hyperphagia, compared with WT mice. Finally, fasting-induced hyperphagia of WT mice was significantly smaller in mice centrally treated with the GHSR inverse agonist K-(D-1-Nal)-FwLL-NH2, compared with mice treated with vehicle, whereas it was unaffected in mice centrally treated with the GHSR antagonists D-Lys3-growth hormone-releasing peptide 6 or JMV2959. Taken together, genetic models and pharmacological results support the notion that constitutive GHSR activity modulates the magnitude of the compensatory hyperphagia triggered by fasting. Thus, the hypothalamic GHSR signaling system could affect the set point of daily food intake, independently of plasma ghrelin levels, in situations of negative energy balance., Instituto Multidisciplinario de Biología Celular

Breast cancer (BC) consists of multiple subtypes defined by various molecular characteristics, for instance, estrogen receptor (ER) expression. Methods for visualizing BC include mammography, MR imaging, ultrasound, and nuclear medicine-based methods such as (99m)Tcsestamibi and F-18-FDG PET, unfortunately all lacking specificity. Peptide receptor scintigraphy and peptide receptor radionuclide therapy are successfully applied for imaging and therapy of somatostatin receptor-expressing neuroendocrine tumors using somatostatin receptor radioligands. On the basis of a similar rationale, radioligands targeting the gastrin-releasing peptide receptor (GRP-R) might offer a specific method for imaging and therapy of BC. The aim of this study was to explore the application of GRP-R radioligands for imaging and therapy of BC by introducing valid preclinical in vitro and in vivo models. Methods: GRP-R expression of 50 clinical BC specimens and the correlation with ER expression was studied by in vitro autoradiography with the GRP-R agonist In-111-AMBA. GRP-R expression was also analyzed in 9 BC cell lines applying (11)1In-AMBA internalization assays and quantitative reverse transcriptase polymerase chain reaction. In vitro cytotoxicity of Lu-177-AMBA was determined on the GRP-Rexpressing BC cell line T47D. SPECT/CT imaging and biodistribution were studied in mice with subcutaneous and orthotopic ER-positive T47D and MCF7 xenografts after injection of the GRP-R antagonist In-111-JMV4168. Results: Most of the human BC specimens (96%) and BC cell lines (6/9) were found to express GRP-R. GRP-R tumor expression was positively (P = 0.026, chi(2)(4) = 12,911) correlated with ER expression in the human BC specimens. Treatment of T47D cells with 10(-7) M/50 MBq of Lu-177-AMBA resulted in 80% reduction of cells in vitro. Furthermore, subcutaneous and orthotopic tumors from both BC cell lines were successfully visualized in vivo by SPECT/CT using In-111-JMV4168; T47D tumors exhibited a higher uptake than MCF7 xenografts. Conclusion: Targeting GRP-R-expressing BC tumors using GRP-R radioligands is promising for nuclear imaging and therapy, especially in ER-positive BC patients.

International audience; VGF is a propeptide of 617 amino acids expressed throughout the central and the peripheral nervous system. VGF and peptides derived from its processing have been found in dense core vesicles and are released from neuronal and neuroendocrine cells via the regulated secretory pathway. Among VGF-derived neuropeptides, TLQP-21 (VGF556-576) has raised a huge interest and is one of most studied. TLQP-21 is a multifunctional neuropeptide involved in the control of several physiological functions, potentially including energy homeostasis, pain modulation, stress responsiveness and reproduction. Although little information is available about its receptor and the intracellular mechanisms mediating its biological effects, recent reports suggest that TLQP-21 may bind to the complement receptors C3aR1 and/or gC1qR. The first aim of this study was to ascertain the existence and nature of TLQP-21 binding sites in CHO cells. Secondly, we endeavored to characterize the ligand binding to these sites by using a small panel of VGF-derived peptides. And finally, we investigated the influence of TLQP-21 on selected intracellular signaling pathways. We report that CHO cells express a single class of saturable and specific binding sites for TLQP-21 with an affinity and capacity of Kd = 0.55 ± 0.05 × 10-9 M and Bmax = 81.7 ± 3.9 fmol/mg protein, respectively. Among the many bioactive products derived from the C-terminal region of VGF that we tested, TLQP-21 was the most potent in stimulating intracellular calcium mobilization in CHO cells; this effect is primarily due to its C-terminal fragment (HFHH-10). TLQP-21 induced rapid and transient dephosphorylation of phospholipase Cγ1 and phospholipase A2. Generation of IP3 and diacylglycerol was crucial for TLQP-21 bioactivity. In conclusion, our results suggest that the receptor stimulated by TLQP-21 belongs to the family of the Gq-coupled receptors, and its activation first increases membrane-lipid derived second messengers which thereby induce the mobilization of Ca2+ from the endoplasmic reticulum followed by a slower store-operated Ca2+ entry from outside the cell.

International audience; TLQP-21 (TLQPPASSRRRHFHHALPPAR) is a multifunctional peptide that is involved in the control of physiological functions, including feeding, reproduction, stress responsiveness, and general homeostasis. Despite the huge interest in TLQP-21 biological activity, very little is known about its intracellular mechanisms of action. In microglial cells, TLQP-21 stimulates increases of intracellular Ca2+ that may activate functions, including proliferation, migration, phagocytosis and production of inflammatory molecules. Our aim was to investigate whether JMV5656 (RRRHFHHALPPAR), a novel short analogue of TLQP-21, stimulates intracellular Ca2+ in the N9 microglia cells, and whether this Ca2+ elevation is coupled with the activation Ca2+-sensitive K+ channels. TLQP-21 and JMV5656 induced a sharp, dose-dependent increment in intracellular calcium. In 77% of cells, JMV5656 also caused an increase in the total outward currents, which was blunted by TEA (tetraethyl ammonium chloride), a non-selective blocker of voltage-dependent and Ca2+-activated potassium (K+) channels. Moreover, the effects of ion channel blockers charybdotoxin and iberiotoxin, suggested that multiple calcium-activated K+ channel types drove the outward current stimulated by JMV5656. Additionally, inhibition of JMV5656-stimulated outward currents by NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4 benzothiazin-3(4H)-one) and TRAM-34 (triarylmethane-34), indicated that KCa3.1 channels are involved in this JMV5656 mechanisms of action. In summary, we demonstrate that, in N9 microglia cells, the interaction of JMV5656 with the TLQP-21 receptors induced an increase in intracellular Ca2+, and, following extracellular Ca2+ entry, the opening of KCa3.1 channels.

Gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer and is being used as a target for molecular imaging. In this study, we report on the direct comparison of 3 novel GRPR-targeted radiolabeled tracers: (AlF)-F-18-JMV5132, Ga-68-JMV5132, and Ga-68-JMV4168 (JMV5132 is NODA-MPAA-beta Ala-beta Ala-[H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], JMV4168 is DOTA-beta Ala-beta Ala-[H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], and NODA-MPAA is 2-[4-(carboxymethyl)-7-{[4-(carboxymethyl) phenyl]methyl}-1,4,7-triazacyclononan-1-yl] acetic acid). Methods: The GRPR antagonist JMV594 (H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) was conjugated to NODA-MPAA for labeling with (AlF)-F-18. JMV5132 was radiolabeled with Ga-68 and F-18, and JMV4168 was labeled with Ga-68 for comparison. The inhibitory concentration of 50% values for binding GRPR of JMV4168, JMV5132, Ga-nat-JMV4168, and Ga-nat-JMV5132 were determined in a competition-binding assay using GRPR-overexpressing PC-3 tumors. The tumor-targeting characteristics of the compounds were assessed in mice bearing subcutaneous PC-3 xenografts. Small-animal PET/CT images were acquired, and tracer biodistribution was determined by ex vivo measurements. Results: JMV5132 was labeled with F-18 in a novel 1-pot, 1-step procedure within 20 min, without need for further purification and resulting in a specific activity of 35 MBq/nmol. Inhibitory concentration of 50% values (in nM) for GRPR binding of JMV5132, JMV4168, Ga-nat-JMV5132, Ga-nat-JMV4168, and (AlF)-F-nat-JMV5132 were 6.8 (95% confidence intervals [CIs], 4.6-10.0), 13.2 (95% CIs, 5.9-29.3), 3.0 (95% CIs, 1.5-6.0), 3.2 (95% CIs, 1.8-5.9), and 10.0 (95% CIs, 6.3-16.0), respectively. In mice with subcutaneous PC-3 xenografts, all tracers cleared rapidly from the blood, exclusively via the kidneys for Ga-68-JMV4168 and partially hepatobiliary for Ga-68-JMV5132 and (AlF)-F-18-JMV5132. Two hours after injection, the uptake of Ga-68-JMV4168, Ga-68-JMV5132, and (AlF)-F-18-JMV5132 in PC-3 tumors was 5.96 +/- 1.39, 5.24 +/- 0.29, 5.30 +/- 0.98 (percentage injected dose per gram), respectively. GRPR specificity was confirmed by significantly reduced tumor uptake of the 3 tracers after coinjection of a 100-fold excess of unlabeled JMV4168 or JMV5132. Small-animal PET/CT clearly visualized PC-3 tumors, with the highest resolution observed for Al18F-JMV5132. Conclusion: JMV5132 could be rapidly and efficiently labeled with F-18. (AlF)-F-18-JMV5132, Ga-68-JMV5132, and Ga-68-JMV4168 all showed comparable high and specific accumulation in GRPR-positive PC-3 tumors. These new PET tracers are promising candidates for future clinical translation.

Ghrelin has anticonvulsant and neuroprotective effects in models of chemoconvulsant-induced seizures and status epilepticus. In this study we investigated whether deletion of the ghrelin receptor could alter the kindling process in the 6 Hz corneal kindling model and whether ghrelin receptor ligands possess anticonvulsant effects in fully kindled mice. Ghrelin receptor wild-type and knockout mice were electrically stimulated at a subconvulsive current twice daily via corneal electrodes until they reached the fully kindled state. Mice lacking the ghrelin receptor showed similar seizure severity during kindling acquisition as well as in the maintenance phase when compared to their wild-type littermates. Subsequently we proceeded by investigating possible anticonvulsant effects of the ghrelin receptor ligands in the acute 6 Hz seizure model and the fully 6 Hz kindled mice. The ghrelin receptor agonist JMV-1843 decreased the seizure severity score both in acutely 6 Hz stimulated mice and in fully kindled ghrelin receptor wild-type mice, but not in fully kindled ghrelin receptor knockout mice. No effect on seizure severity was observed following the ghrelin receptor antagonist JMV-2959 in both models. This finding indicates that JMV-1843 exerts an anticonvulsant effect in kindled mice via the ghrelin receptor.

We developed a simple and straightforward way to dimerize unprotected peptide sequences that relies on a chemoselective condensation of hybrid peptides bearing a hydroxydimethylsilyl group at a chosen position (either C-ter, N-ter or side-chain linked) to generate siloxane bonds upon freeze-drying. Interestingly, the siloxane bond sensitivity to hydrolysis is strongly pH-dependent. Thus, we investigated the stability of siloxane dimers in different experimental conditions. For that purpose, 29Si, 13C and 1H NMR spectra were recorded to accurately quantify the ratio of dimer/monomer. More interestingly, we showed that 1H resonances of the methylene and methyl groups connected to the Si can be used as sensitive probes to monitor siloxane hydrolysis and to determine the half-lives of the dimers. Importantly, we showed that the dimers were rather stable at pH 7.4 (t1/2 ≈ 400 h) and we applied the dimerization strategy to bioactive sequences. Once optimized, three dimers of the growth hormone releasing hexapeptide (GHRP-6) were prepared. Interestingly, their pharmacological evaluation revealed that the activity of the dimeric ligands could be switched from agonist to inverse agonist depending on the position of dimerization.

The 6-Hz corneal stimulation test is used to screen novel antiepileptic molecules to overcome the problem of drug refractoriness. Although recognized as a standard test, it has been evaluated only recently in the attempt to characterize the putative neuronal networks involved in seizures caused by corneal stimulation. In particular, by recording from the CA1 region we previously established that the hippocampus participates to propagation of seizure activity. However, these findings were not corroborated by using markers of neuronal activation such as FosB/ΔFosB antigens. In view of this discrepancy, we performed new experiments to characterize the changes in levels of phosphorylated extracellular signal-regulated kinases1/2 (p-ERK1/2), which are also used as markers of neuronal activation. To this aim, mice underwent corneal stimulation up to three different times, in three sessions separated by an interval of 3 days. To characterize a group in which seizures could be prevented by pharmacological treatment, we also considered pretreatment with the ghrelin receptor antagonist EP-80317 (330 μg/kg). Control mice were sham-treated. Video electrocorticographic (ECoG) recordings were obtained from mice belonging to each group of treatment. Animals were finally used to characterize the immunoreactivity for FosB/ΔFosB and p-ERK1/2 in the hippocampus. As previously shown, FosB/ΔFosB levels were highly increased throughout the hippocampus by the first induced seizure but, in spite of the progressively increased seizure severity, they were restored to control levels after the third stimulation. At variance, corneal stimulation caused a progressive increase in p-ERK1/2 immunoreactivity all over the hippocampus, especially in CA1, peaking in the third session. Predictably, EP-80317 administration reduced both duration and severity of seizures, prevented the increase in FosB/ΔFosB levels in the first session, and partially counteracted the increase in p-ERK1/2 levels in the third session. The vast majority of p-ERK1/2 immunopositive cells were co-labeled with FosB/ΔFosB antibodies, suggesting the existence of a relationship between the investigated markers in a subpopulation of neurons activated by seizures. These findings suggest that p-ERK1/2 are useful markers to define the aggravation of seizures and the response to anticonvulsant treatments. In particular, p-ERK1/2 expression clearly identified the involvement of hippocampal regions during seizure aggravation in the 6-Hz model.

Preparative HPLC fractionation curve of the raw extract from S. pobeguinii bark (E10). (PDF 189 kb)

HPTLC of extracts 10, 14 and 15 using DPPH derivatization reagent. The DPPH test shows some very prominent antioxidant zones. (PDF 17 kb)

Diagram showing inhibition of α-glucosidase by extracts 14 and 15 at three concentrations. α-Glucosidase was incubated with extracts at a concentration of 1, 0.1 and 0.01 mg/mL. Remaining activities in the presence of extracts are expressed as a percentage of enzyme activity incubated in DMSO alone and are the mean ± SE of three separate determinations. 10 mM Acarbose was used as specific inhibitor of α-glucosidase. (PDF 30 kb)

Preparative HPLC fractionation curve of the raw extract from N. diderrichii leaves (E14). (PDF 207 kb)

International audience; A single tool for early detection, accurate staging, and personalized treatment of prostate cancer (PCa) would be a major breakthrough in the field of PCa. Gastrin-releasing peptide receptor (GRPR) targeting peptides are promising probes for a theranostic approach for PCa overexpressing GRPR. However, the successful application of small peptides in a theranostic approach is often hampered by their fast in vivo degradation by proteolytic enzymes, such as neutral endopeptidase (NEP). Here we show for the first time that co-injection of a NEP inhibitor (phosphoramidon (PA)) can lead to an impressive enhancement of diagnostic sensitivity and therapeutic efficacy of the theranostic 68Ga-/177Lu-JMV4168 GRPR-antagonist. Co-injection of PA (300 µg) led to stabilization of 177Lu-JMV4168 in murine peripheral blood. In PC-3 tumor-bearing mice, PA co-injection led to a two-fold increase in tumor uptake of 68Ga-/177Lu-JMV4168, 1 h after injection. In positron emission tomography (PET) imaging with 68Ga-JMV4168, PA co-injection substantially enhanced PC-3 tumor signal intensity. Radionuclide therapy with 177Lu-JMV4168 resulted in significant regression of PC-3 tumor size. Radionuclide therapy efficacy was confirmed by production of DNA double strand breaks, decreased cell proliferation and increased apoptosis. Increased survival rates were observed in mice treated with 177Lu-JMV4168 plus PA as compared to those without PA. This data shows that co-injection of the enzyme inhibitor PA greatly enhances the theranostic potential of GRPR-radioantagonists for future application in PCa patients.

Diagram showing inhibition of β-glucosidase by extracts 14 and 15 at three concentrations. β-Glucosidase was incubated with extracts at a concentration of 1, 0.1 and 0.01 mg/mL. Remaining activities in the presence of extracts are expressed as a percentage of enzyme activity incubated in DMSO alone and are the mean ± SE of three separate determinations. 1 μM Isofagomine was used as specific inhibitor of β-glucosidase. (PDF 30 kb)

International audience; The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, μ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.

Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored. In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, constitutive activity. To identify the molecular mechanisms responsible for this high constitutive activity, we have reconstituted a purified GHS-R1a monomer in a lipid disc. Using this reconstituted system, we show that the isolated ghrelin receptor per se activates Gq in the absence of agonist, as assessed through guanosine 5′-O-(thiotriphosphate) binding experiments. The measured constitutive activity is similar in its extent to that observed in heterologous systems and in vivo. This is the first direct evidence for the high constitutive activity of the ghrelin receptor being an intrinsic property of the protein rather than the result of influence of its cellular environment. Moreover, we show that the isolated receptor in lipid discs recruits arrestin-2 in an agonist-dependent manner, whereas it interacts with μ-AP2 in the absence of ligand or in the presence of ghrelin. Of importance, these differences are linked to ligand-specific GHS-R1a conformations, as assessed by intrinsic fluorescence measurements. The distinct ligand requirements for the interaction of purified GHS-R1a with arrestin and AP2 provide a new rationale to the differences in basal and agonist-induced internalization observed in cells.

International audience; Aims: Ghrelin (GHR) is an orexigenic gut peptide that interacts with ghrelin receptors (GHR-Rs) to modulate brain reinforcement circuits. Systemic GHR infusions augment cocaine stimulated locomotion and conditioned place preference (CPP) in rats, whereas genetic or pharmacological ablation of GHR-Rs has been shown to attenuate the acute locomotor-enhancing effects of nicotine, cocaine, amphetamine and alcohol and to blunt the CPP induced by food, alcohol, amphetamine and cocaine in mice. The stimulant nicotine can induce CPP and like amphetamine and cocaine, repeated administration of nicotine induces locomotor sensitization in rats. A key issue is whether pharmacological antagonismof GHR-Rs would similarly attenuate nicotine-induced locomotor sensitization. Method: To examine the role of GHR-Rs in the behavioral sensitizing effects of nicotine, adult male rats were injected with either 0, 3 or 6 mg/kg of the GHR-R receptor antagonist JMV 2959 (i.p.) and 20 min later with either vehicle or 0.4 mg/kg nicotine hydrogen tartrate (s.c.) on each of 7 consecutive days. Results: Rats treated with nicotine alone showed robust locomotor sensitization, whereas rats pretreated with JMV 2959 showed significantly attenuated nicotine-induced hyperlocomotion. Conclusions: These results suggest that GHR-R activity is required for the induction of locomotor sensitization to nicotine and complement an emerging literature implicating central GHR systems in drug reward/reinforcement.

Systemic infusions of the orexigenic peptide ghrelin (GHR) increase dopamine levels within the nucleus accumbens and augment cocaine-stimulated locomotion and conditioned place preference in rats; observations that suggest an important role for GHR and GHR receptors (GHR-Rs) in drug reinforcement. In the present studies, we examined the development of cocaine locomotor sensitization in rats, sustaining either pharmacologic antagonism or genetic ablation of GHR-Rs. In a pharmacologic study, adult male rats were injected (i.p.) with either 0, 3 or 6 mg/kg JMV 2959 (a GHR-R1 receptor antagonist), and 20 minutes later, with either vehicle or 10 mg/kg cocaine HCl on each of 7 consecutive days. Rats pretreated with JMV 2959 showed significantly attenuated cocaine-induced hyperlocomotion. In a second study, adult wild-type (WT) or mutant rats sustaining ENU-induced knockout of GHR-R [GHR-R ((-/-) )] received daily injections (i.p.) of vehicle (0.9% saline) or 10.0 mg/kg cocaine HCl for 14 successive days. GHR-R null rats treated repeatedly with cocaine showed diminished development of cocaine locomotor sensitization relative to WT rats treated with cocaine. To verify the lack of GHR-R function in the GHR-R ((-/-) ) rats, a separate feeding experiment was conducted in which WT rats, but not GHR-R ((-/-) ) rats, were noted to eat more after a systemic injection of 15 nmol GHR than after vehicle. These results suggest that GHR-R activity is required for the induction of locomotor sensitization to cocaine and complement an emerging literature implicating central GHR systems in drug reward. GHR is an orexigenic gut peptide that is transported across the blood-brain barrier and interacts with GHR-Rs located on ventral tegmental dopamine neurons.

The knowledge of the binding sites of G protein-coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin-A receptor (CCK-AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three-dimensional model of the [CCK-AR-CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site-directed mutagenesis. Wild-type and mutated CCK-AR were transiently expressed in COS-7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I-BH-[Thr, Nle]-CCK-9; however, it bound the nonpeptide antagonist [3H]-SR27,897 as the wild-type receptor. The mutant was approximately 1,470- and 3,200-fold less potent than the wild-type CCK-AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500-fold lower potency and 800-fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild-type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK-AR, strongly support an interaction between Arg197 of the CCK-AR binding site and the sulfate of CCK. In addition, the mutated CCK-AR resembled the low affinity state of the wild-type CCK-AR, suggesting that Arg197-sulfate interaction regulates conformational changes of the CCK-AR that are required for its physiological activation.

International audience; The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides β-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger β-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.

International audience; Feeding behavior is exquisitely regulated by homeostatic and hedonic neural substrates that integrate energy demand as well as the reinforcing and rewarding aspects of food. Understanding the net contribution of homeostatic and reward-driven feeding has become critical because of the ubiquitous source of energy-dense foods and the consequent obesity epidemic. Hypothalamic agouti-related peptide-secreting neurons (AgRP neurons) provide the primary orexigenic drive of homeostatic feeding. Using models of neuronal inhibition or ablation, we demonstrate that the feeding response to a fast ghrelin or serotonin receptor agonist relies on AgRP neurons. However, when palatable food is provided, AgRP neurons are dispensable for an appropriate feeding response. In addition, AgRP-ablated mice present exacerbated stress-induced anorexia and palatable food intake--a hallmark of comfort feeding. These results suggest that, when AgRP neuron activity is impaired, neural circuits sensitive to emotion and stress are engaged and modulated by food palatability and dopamine signaling.

International audience; How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G proteinactivation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor: G protein complex, ultimately leading to G protein activation and signaling.

1 The growth hormone secretagogue receptor 1a (GHSR-1a) is a G-protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44- and 42-kDa extracellular signal-regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR-1a dependent or not. 2 To provide direct evidence for the coupling of GHSR-1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3 Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR-1a, a transient and dose-dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4 We then investigated the precise mechanisms involved in GHSR-1a-mediated ERK1/2 activation using various specific inhibitors and dominant-negative mutants and found that internalization of GHSR-1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR-1a-mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3-kinases were not found to be involved. GHSR-1a-mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Go6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA-AM, and the inhibitor of conventional PKCs, Go6976, had no effect on the GHSR-1a-mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (ɛ, δ), but not conventional or atypical PKCs. Further analyses suggest that PKCɛ is required for the activation of ERK1/2. 5 Taken together, these data suggest that ghrelin, through GHSR-1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC- and PKCɛ-dependent pathway. British Journal of Pharmacology (2006) 148, 350–365. doi:10.1038/sj.bjp.0706727