Franco Barrile

Céline M'kadmi

Pablo N De Francesco

Agustina Cabral

Guadalupe García Romero

Emilio R Mustafá

Sonia Cantel

Marjorie Damian

Sophie Mary

Séverine Denoyelle

Jean-Louis Banères

Jacky Marie

Jesica Raingo

Jean-Alain Fehrentz
email: jean-alain.fehrentz@umontpellier.fr.-

Mario Perelló
email: mperello@imbice.gov.ar.

Development of a novel fluorescent ligand of growth hormone secretagogue receptor based on the N-Terminal Leap2 region

Keywords: ghrelin, G-protein coupled receptor, inverse agonist, fluorescent probe

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The growth hormone secretagogue receptor (GHSR) is a prototypical class A G protein coupled receptor (GPCR) [START_REF] Howard | A receptor in pituitary and hypothalamus that functions in growth hormone release[END_REF]. Ghrelin, a gastrointestinal tract-derived octanoylated 28-residue peptide hormone, was the first known endogenous ligand for GHSR [START_REF] Kojima | Ghrelin is a growth-hormone-releasing acylated peptide from stomach[END_REF]. Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as another endogenous ligand for GHSR [START_REF] Ge | LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor[END_REF]. Ghrelin activates GHSR signaling, while LEAP2 acts as both antagonist and inverse agonist of GHSR [START_REF] Ge | LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor[END_REF][START_REF] M'kadmi | N-Terminal Liver-Expressed Antimicrobial Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the Ghrelin Receptor[END_REF][START_REF] Wang | Identifying the binding mechanism of LEAP2 to receptor GHSR1a[END_REF]. GHSR also exerts ligand-independent actions as it displays a high constitutive activity, which reaches ~50% of its maximal activity, and can heterodimerize with other GPCRs in order to affect their activity [START_REF] Kern | Hippocampal Dopamine/DRD1 Signaling Dependent on the Ghrelin Receptor[END_REF][START_REF] Kern | Apo-ghrelin receptor forms heteromers with DRD2 in hypothalamic neurons and is essential for anorexigenic effects of DRD2 agonism[END_REF][START_REF] Schellekens | Ghrelin's Orexigenic Effect Is Modulated via a Serotonin 2C Receptor Interaction[END_REF]. GHSR regulates key physiological functions in humans. For instance, up-regulation of GHSR signaling potently increases growth hormone (GH) secretion and promotes mechanisms that increase glycaemia [START_REF] Garin | Clinical review: The human experience with ghrelin administration[END_REF]. Ghrelin-induced activation of GHSR also potently and rapidly stimulates food intake [START_REF] Garin | Clinical review: The human experience with ghrelin administration[END_REF]. Notably, GHSR is highly expressed in the brain and, consequently, regulates a variety of central functions including reward-related cognition, associative learning, memory, stress responses, among others [START_REF] Morin | The effects of ghrelin on sleep, appetite, and memory, and its possible role in depression: A review of the literature[END_REF][START_REF] Perello | Ghrelin signalling on food reward: a salient link between the gut and the mesolimbic system[END_REF]. Thus, GHSR is a highly attractive drug target to treat a variety of pathological conditions in human beings.

Development of fluorescent GPCR ligands is a major topic in pharmacology for in vitro and in vivo applications [START_REF] Iliopoulos-Tsoutsouvas | Fluorescent probes for G-protein-coupled receptor drug discovery[END_REF]. The use of fluorescently-labeled ghrelin was essential to study the localization, function and regulation of GHSR. In particular, we reported the first fluorescence-based ligand binding assay for GHSR relying on fluorescence resonance energy transfer (FRET) between fluorescent versions of GHSR and ghrelin [START_REF] Degorce | HTRF: A technology tailored for drug discovery -a review of theoretical aspects and recent applications[END_REF][START_REF] Keppler | Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro[END_REF][START_REF] Leyris | Homogeneous time-resolved fluorescencebased assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a[END_REF][START_REF] Maurel | Cell-surface proteinprotein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization[END_REF]. We also used fluorescent variants of ghrelin to gain insights about different aspects of the physiology or cell biology of GHSR, including its neuroanatomical distribution in the mouse brain [START_REF] Cabral | Analysis of brain nuclei accessible to ghrelin present in the cerebrospinal fluid[END_REF][START_REF] Cornejo | Ghrelin receptor signaling targets segregated clusters of neurons within the nucleus of the solitary tract[END_REF][START_REF] Fernandez | Des-Acyl Ghrelin Directly Targets the Arcuate Nucleus in a Ghrelin-Receptor Independent Manner and Impairs the Orexigenic Effect of Ghrelin[END_REF], its regulation by changes in the energy balance [START_REF] Fernandez | Evidence Supporting a Role for Constitutive Ghrelin Receptor Signaling in Fasting-Induced Hyperphagia in Male Mice[END_REF], its subcellular localization in neurons [START_REF] Cabral | Ghrelin activates hypophysiotropic corticotropin-releasing factor neurons independently of the arcuate nucleus[END_REF], its cellular trafficking in in vitro systems [START_REF] Soto | Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons[END_REF] and its ability to interact with plasma proteins and other GPCRs [START_REF] Lufrano | Ghrelin binding to serum albumin and its biological impact[END_REF][START_REF] Schellekens | Ghrelin's Orexigenic Effect Is Modulated via a Serotonin 2C Receptor Interaction[END_REF]. In addition, fluorescently-labeled ghrelin was instrumental to clarify the mechanisms transporting ghrelin into the brain [START_REF] Cabral | Circulating Ghrelin Acts on GABA Neurons of the Area Postrema and Mediates Gastric Emptying in Male Mice[END_REF][START_REF] Cabral | Divergent neuronal circuitries underlying acute orexigenic effects of peripheral or central ghrelin: critical role of brain accessibility[END_REF][START_REF] Schaeffer | Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons[END_REF][START_REF] Uriarte | Evidence Supporting a Role for the Blood-Cerebrospinal Fluid Barrier Transporting Circulating Ghrelin into the Brain[END_REF]. Still, ghrelin-derived fluorescent probes have some limitations as tools to investigate the biology of GHSR. First, ghrelin half-life in vivo is very short because the ester bond that links the n-octanoyl moiety to serine 3 is susceptible to be hydrolyzed either spontaneously or by plasma esterases, and des-acyl ghrelin displays a very poor affinity for GHSR [START_REF] De Vriese | Ghrelin interacts with human plasma lipoproteins[END_REF][START_REF] Hosoda | Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue[END_REF]. In order to avoid this limitation, serine 3 of ghrelin sequence can be replaced by either 3,4-diaminopropionic acid or aspartic acid, which are then linked through an amide bond with octanoic acid or octylamine, respectively [START_REF] Leyris | Homogeneous time-resolved fluorescencebased assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a[END_REF][START_REF] Mcgirr | Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a[END_REF]. These modifications appear to have a minimal impact on the interaction of the probes with GHSR [START_REF] Leyris | Homogeneous time-resolved fluorescencebased assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a[END_REF][START_REF] Mcgirr | Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a[END_REF]; however, they add an extra level of complexity in their biosynthesis. Another feature regarding the use of fluorescentlylabeled ghrelin to investigate the biology of GHSR is the ability of ghrelin to promote GHSR internalization and recycling in living cells [START_REF] Camiña | Desensitization and endocytosis mechanisms of ghrelin-activated growth hormone secretagogue receptor 1a[END_REF]. These cellular mechanisms may interfere with the accurate estimation of FRET signal or reduce the sensitivity of fluorescent assays in some experimental designs. Finally, development of fluorescent probes with a pharmacological profile different of the natural agonist ligand would be useful for the analysis of ligand binding or dimerization properties of GHSR. Recently, we established that LEAP2 binds to the orthosteric site of GHSR and that the N-terminal part of LEAP2 is sufficient to bind to GHSR and reduce its constitutive activity [START_REF] M'kadmi | N-Terminal Liver-Expressed Antimicrobial Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the Ghrelin Receptor[END_REF]. Importantly, LEAP2 should not promote GHSR internalization due to its pharmacological properties and, as a consequence, could label GHSR without any effect on its localization. Thus, we decided to develop a new fluorescent GHSR ligand based on the N-terminal sequence of LEAP2 that should prove of paramount interest to characterize the molecular behavior of GHSR and to investigate not only the physiology and cell biology of GHSR but also specific aspects inherent to the biology of LEAP2.

MATHERIAL AND METHODS

F-LEAP2 synthesis.

H-1 Nle-2 Thr-3 Pro-4 Phe- 5 Trp-6 Arg- 7 Gly-8 Val-9 Ser-10 Leu-11 Arg-12 Pro-13 Ile-14 Gly-15 Ala-16 Ser-17 Cys-NH2 was synthesized by solid-phase peptide synthesis starting from Agilent Amphisphere 40 RAM resin using Fmoc chemistry, HATU/DIEA system for coupling and piperidine/DMF for deprotection. All coupling steps (5 eq.) were performed twice for 10 min, with the exception of Fmoc-Thr 2 (tBu)-OH which was coupled twice for 15 min. Final deprotection was performed with a TFA/TIS/H2O (95/2.5/2.5) mixture for 3 h. After purification by preparative RP-HPLC, the peptide (0.845 eq.) was dissolved in 1 mL of sodium phosphate solution (pH 7) and conjugated with 1 mg of DY-647P1-maleimide (Dyomics, Jena, Germany) for 3 h. The fluorescent peptide was directly injected on a preparative RP-HPLC column and purified. Their identity and purity were evaluated by mass spectrometry analyses. Preparative RP-HPLC was run on a Gilson PLC 2250 Purification system (Villiers le Bel, France) instrument using a preparative column (Waters DeltaPak C18 Radial-Pak Cartridge, 100 Å, 40-100 mm, 15 µm particle size) in gradient mode with a flow rate 50.0 mL/min. Buffer A was 0.1% TFA in water, and buffer B was 0.1% TFA in acetonitrile.

LC/MS analyses.

The LC/MS system consisted of a HPLC-ZQ (Waters) or UPLC Acquity H-Class (Waters) coupled to a Synapt G2-S (Waters) equipped with an ESI source. Analyses were carried out using a Phenomenex Kinetex column (C18, 100 Å, 100x2.1 mm, 2.6µm). A flow rate of 0.5 mL/min and a gradient of 0-100% B in 5 min were used: eluent A, water/0.1% HCO2H; eluent B, ACN/0.1% HCO2H. Positive-ion electrospray (ESI+) mass spectra were acquired from 100 to 1500 m/z with a scan time of 0.2 s. Nitrogen was used for both the nebulizing and drying gas.

MALDI MS and MS/MS Analyses.

Samples were analyzed from CHCA or SA matrix deposits, in positive ion mode the Rapiflex instrument from Bruker Daltonics © . A pulsed Nd:YAG laser at a wavelength of 355 nm was operated at a 66.7 Hz frequency with a laser focus of 29%. Data were acquired with the Flex Control software (version 4.1, Bruker Daltonics © ). Spectra were integrated with the Flex Analysis software (version 4.0, BrukerDaltonics © ), the centroid algorithm was used to assign peaks. An acceleration voltage of 25.0 kV (IS1) was applied for a final acceleration of 21.95 kV (IS2) and a lense voltage of 9.6 kV. The reflectron mode was used for the ToF analyser (voltages of 26.3 kV and 13.8 kV). The delayed extraction time was 30 ns. Acquisitions were performed using a reflector detector voltage of 1.722 kV. MS data were processed with the Flex Analysis software (version 4.0, Bruker Daltonics © ). External calibration was performed with commercial peptide mixture (Calibration peptide standard II, protein I, Bruker Daltonics, Wissembourg, France).

Fragmentation experiments were performed under laser induced dissociation (LID) conditions with the LIFT cell voltage parameters set at 19.0 kV (LIFT 1) and 3.7 kV (LIFT 2) for a final acceleration of 29.5 kV (reflector voltage) and a pressure in the LIFT cell around 4 x 10 -7 mbar. The precursor ion selector was set manually to the first monoisotopic peak of the molecular ion pattern for all analyses. MS/MS data were processed with the Flex Analysis software (version 4.0, Bruker Daltonics © ). Mass lists were generated according to the following parameters: SNAP as peak detection algorithm, S/N threshold 3.

Drugs.

Ghrelin was purchased from Global Peptides (cat# PI-G-03). Full-length LEAP2 and LEAP2(1-14)-NH2 were synthesized, purified by RP-HPLC and characterized by LC-MS and MALDI-MS/MS with a purity> 95%, as described previously (M' Kadmi et al., 2019). Fluorescent ghrelin (hereafter named F-ghrelin) was a variant of ghrelin conjugated to DY-647P1 fluorophore through a C-terminal Cys, as described above for F-LEAP2. Specifically, F-ghrelin was synthesized by linking DY-647P1-maleimide to a 19-residues ghrelin analog that contains the initial 18 residues of the ghrelin sequence, but replacing the serine in position 3 by aspartic acid whose side-chain was amidated with amino-octanoic acid, and cysteine at the position 19. F-ghrelin was purified by RP-HPLC and characterized by LC-MS and MALDI-MS/MS with a purity> 95%, as described above for F-LEAP2.

Saturation binding assay.

This assay is based on the Tag-lite technology that combines homogeneous time-resolved fluorescence (HTRF) detection with a covalent labeling technology called SNAP-tag [START_REF] Degorce | HTRF: A technology tailored for drug discovery -a review of theoretical aspects and recent applications[END_REF][START_REF] Keppler | Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro[END_REF][START_REF] Maurel | Cell-surface proteinprotein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization[END_REF]. Specifically, F-LEAP2 was used as HTRF acceptor, and GHSR fused with the SNAP-tag enzyme (SNAP-GHSR) and covalently labeled with a terbium cryptate (Lumi4-Tb) was used as a HTRF donor [START_REF] Leyris | Homogeneous time-resolved fluorescencebased assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a[END_REF]. HEK293T cells expressing the SNAP-GHSR were incubated with increasing concentrations of F-LEAP2 diluted in the Tag-lite labelling medium. For each concentration, nonspecific binding was determined by adding 5 µM unlabeled LEAP2 diluted in the same buffer. Plates were incubated for 1 h at room temperature. Two independent experiments were performed, each of them in triplicate.

In vitro assessment of inverse agonist properties of F-LEAP2.

Monomeric GHSR in lipid nanodiscs and Gαqβ1γ2 trimer were prepared as previously described [START_REF] Damian | GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation[END_REF][START_REF] Damian | High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs[END_REF]. GTPγS binding experiments were carried out using a fluorescent BODIPY-FL GTPγS analog [START_REF] Mcewen | Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins[END_REF]. Association of BODIPY-FL GTPγS to the G protein was monitored using a fluorescence spectrophotometer (Cary Eclipse, Varian) with the excitation wavelength set at 500 nm and the emission wavelength at 511 nm. Reaction conditions were 100 nM Gαq, 500 nM Gβ1γ2, 100 nM BODIPY-FL GTPγS, and 20 nM GHSR in lipid discs. GHSR, the ligands (ghrelin, LEAP2 or F-LEAP2 at a 10 µM final concentration) and Gαqβ1γ2 protein were first incubated for 30 min at room temperature, then BODIPY-FL GTPγS was added and fluorescence was measured after 10 min incubation at 15°C. Three independent experiments were performed. Data were presented as the percentage of maximum BODIPY-FL fluorescence change measured in the presence of ghrelin.

2.7.

Cell culture imaging studies. TsA201 cells were plated on glass coverslips previously treated with poly-L-lysine and laid over 24-well plates. After 24 h, cells growing in Dulbecco's modified Eagle's medium with 10% fetal bovine serum were transfected with GHSR-yellow fluorescent protein (YFP)-expressing plasmid using Lipofectamine 2000. After 24 h, cells were pre-incubated with medium alone or containing F-LEAP2 (15 nM) or F-ghrelin (15 nM) for 15 min. In some cases, cells were pre-incubated with medium alone or containing LEAP2 (5000 nM) or ghrelin (5000 nM) for 2 min and then exposed to medium containing F-LEAP2 (15 nM) for 15 min. After that, cells were rinsed with PBS, fixed with 4% paraformaldehyde in PBS for 30 min, and rinsed again twice with PBS. Finally, glass coverslips with fixed cells were mounted on glass slides using mounting medium containing Hoechst. Fluorescent images were acquired using a Zeiss AxioObserver D1 microscope equipped with an Apotome.2 structured illumination module and an Axiocam 506 monochrome camera. DY-647P1 was excited at 640 nm and detected in the 690 nm range. YFP was excited at 513 nm and detected in the 527 nm range. Hoechst was excited at 352 nm and detected in the 460 nm range. Transfection efficiency was estimated as the ratio between YFP positive cells and the total number of Hoechst positive cells in fluorescent images acquired at low magnification (10X/0.45 objective), and represented ~60% in all the experiments. F-LEAP2 and F-ghrelin signals were estimated in the plasma membrane of YFP positive cells, in fluorescent images acquired at high magnification (63X/1.4 objective) in 12 cells per condition. The binding of the fluorescent analogs to the cell surface was estimated as the difference between the total fluorescent signal at 690 nm of each cell and the internal fluorescent signal at 690 nm in the same cell (excluding plasma membrane). Specifically, the total area of each cell was first delimited by manually tracing the boundaries of the cell using the external limits of the GHSR-YFP fluorescent signal as a reference. Then, the total fluorescence signal intensity at 690 nm was quantified in the delimited area, as integrated density. Next, the delimited area in each cell was concentrically reduced by a fixed amount, in order to exclude the green GHSR-YFP fluorescent signal corresponding to the plasma membrane, and the internal fluorescence signal intensity at 690 nm was quantified in the reduced area as integrated density. Finally, the surface fluorescence signal intensity was calculated by subtracting the internal fluorescence signal from the total fluorescence signal and expressed in arbitrary units (AU). Image analysis was performed in Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF].

Mice.

We used adult (8-12 weeks old) male C57BL/6 wild-type mice that were generated in the animal facility of the IMBICE and housed in a 12-h light/dark cycle with regular chow and water available ad libitum. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and all efforts were made to minimize suffering. All experimentation received approval from the Institutional Animal Care and Use Committees located in Multidisciplinary Institute of Cell Biology (approval ID 17-0221).

2.9.

In vivo labeling and neuroanatomical mapping analysis. Anesthetized mice were implanted with a single indwelling guide intra-cerebroventricular (ICV) cannula (4 mm long, 22 gauge, Plastics One) into the lateral ventricle using a stereotaxic. Placement coordinates were: AP:-0.34 mm; L:+1 mm and V:-2.3 mm. For the in vivo experiment 1 (Figure 1A), mice were ICV-injected with 1 µL of vehicle (artificial cerebrospinal fluid, n=3) or F-LEAP2 (10 pmol, n=7). For the in vivo experiment 2 (Figure 1B), an independent set of mice were first ICV injected with 1 µL of vehicle (n=3), full-length LEAP2 (450 pmol, n=3) or ghrelin (450 pmol, n=3) and immediately after with 1 µL of F-LEAP2 (10 pmol), in order to test the specificity of the F-LEAP2 labeling. ICV injections were made over 2 min through a 30 gauge needle that extend 0.5 mm below the guide cannula. The needle was left in place for 2 min, following the injection, to prevent back flow of the injected solution. After 30 min, anesthetized mice were perfused with formalin as previously described [START_REF] Cabral | Ghrelin indirectly activates hypophysiotropic CRF neurons in rodents[END_REF]. Brains were extracted, post-fixed, immersed in 20% sucrose, and coronally cut at 40 µm into four equal series on a cryostat. Correct placement of the cannula was confirmed by histological observation at the end of the experiment. Brain sections were sequentially mounted on glass slides, coverslipped with mounting medium containing Hoechst, and imaged in a fluorescence microscope using structured illumination, as stated above. The presence of F-LEAP2 signal was determined in 1-in-4 series from the level of the olfactory bulbs down to the cervical spinal cord. Fluorescent images were acquired with a 10X/0.45 objective, using an automated tiling strategy to image all the brain sections. Also, higher magnification images of selected regions were acquired using a 40X/0.95 objective. Tiles from each mouse was aligned for neuroanatomical analysis using the Fiji plugin TrackEM2 [START_REF] Cardona | TrakEM2 software for neural circuit reconstruction[END_REF]. The mouse brain atlas of Paxinos and Franklin [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]was used to identify brain sections and to describe the brain nuclei. Estimates of F-LEAP2 signal for the different brain regions were made by considering both signal strength and number of labeled cells as compared to the signal observed in negative control samples.

Food intake assessment.

For the in vivo experiment 3 (Figure 1C), mice were permanently implanted with an ICV guide cannula by securing the guide cannulas to the skull with cranioplastic cement. After surgery, mice were individually housed and allowed to recover for 5 days. On the morning of the experimental day, ad libitum fed mice were ICV-injected, as described above, with 1 µL of vehicle, full-length LEAP2 (10 pmol) or F-LEAP2 (10 pmol) and immediately after with 1 µL of vehicle or ghrelin (60 pmol, n=8 per experimental group). Then, mice were exposed to a single pre-weighed chow pellet (~1500 mg) in the floor of the home cages and remained undisturbed. Chow pellets were collected after 2 h and weighed. Weighing was performed in calibrated scales that had a precision of 1 mg. Two-hour food intake was calculated subtracting the remaining weight of the pellet to the initial weight, and expressed in mg.

Statistical analyses.

Data were expressed as mean±SEM. Dissociation constant of F-LEAP2 was determined from saturation curves of the specific binding. GTPγS binding experiments and imaging studies were analyzed by ANOVA with Tukey´s post-test. Food intake studies were analyzed by one-way ANOVA test for comparison of different mean values. Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, Inc). Differences were considered statistically significant when p<0.05.

RESULTS AND DISCUSSION

Synthesis of F-LEAP2.

The N-terminal LEAP2 sequence was chosen to develop the fluorescent tracer because it contains the whole bioactive part of the molecule that binds to GHSR. In order to conjugate LEAP2 to a fluorescent moiety, we decided to take advantage of the cysteine 17 , which participates in an internal disulfide bridge with cysteine 28 in the native peptide. Thus, LEAP2(1-17)-NH2 was first synthesized by solid phase and purified. Then, the fluorescent probe DY-647P1 was conjugated with the thiol function of cysteine 17 using DY-647P1-maleimide. The resulting LEAP2(1-17)-DY-647P1 or F-LEAP2 (Figure 2A) was then purified to homogeneity and confirmed to display the correct molecular mass (theoretical value 2669.2 Da; calculated value 2667.3 Da from observed m/z 1335.1

[M+2H] 2+ , m/z 890.3 [M+3H] 3+ , m/z 668.4 [M+4H] 4+ ).
Fragmentation experiments confirmed that F-LEAP2 displayed the correct residue sequence (not shown).

Saturation receptor binding assays for F-LEAP2.

F-LEAP2 binding affinity to GHSR was estimated using a saturation receptor binding assay. As shown in Figure 2B, F-LEAP2 bound the living HEK293T cells transiently overexpressing SNAP-GHSR in a typical, saturable manner, with a calculated dissociation constant of 3.9±1.1 nM. Previously, we found that full-length LEAP2 and LEAP2(1-14)-NH2 displayed dissociation constants of 1.3±0.1 and 3.7±0.6 nM, respectively (M' Kadmi et al., 2019). Thus, F-LEAP2 retained high binding affinity to GHSR despite the attached fluorescent moiety.

In vitro assessment of inverse agonist properties of F-LEAP2.

The ability of F-LEAP2 to affect GHSR-catalyzed Gq activation was tested using GTPγS binding assays. As shown in Figure 2C, GHSR catalyzed GDP-to-GTP exchange on the Gγ subunit of the Gαqβ1γ2 trimer in the absence of ligand, in agreement with its demonstrated high constitutive activity [START_REF] Damian | High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs[END_REF]. As expected, ghrelin further enhanced GPTγS binding. Unlabeled LEAP2(1-14)-NH2 decreased basal GPTγS binding, consistent with the inverse agonist properties of LEAP2 N-terminal region (M' Kadmi et al., 2019). Similarly, F-LEAP2 decreased GPTγS binding, indicating that the fluorophore did not affect the inverse agonist properties of the tracer.

Cell culture imaging studies.

In order to test the ability of F-LEAP2 to label GHSR-expressing cells, GHSR-YFP-expressing cells were incubated with F-LEAP2 and visualized in a fluorescence microscope. As shown in Figure 3, F-LEAP2 exclusively bound to GHSR-YFP-expressing cells and did not bind to nontransfected cells, which lack GHSR-YFP expression. Notably, F-LEAP2 signal was limited to the cell surface. As shown in Figure 3, F-ghrelin also exclusively labeled GHSR-YFP-expressing cells. In contrast to F-LEAP2, F-ghrelin signal was mainly localized inside the GHSR-YFP-expressing cells and with a punctuated pattern that co-localizes with YFP signal. These observations indicate that F-ghrelin induces internalization of GHSR, as previously reported [START_REF] Holliday | Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor[END_REF], while F-LEAP2 does not mediate internalization of the receptor. As also shown in Figure 3, pre-incubation of GHSR-YFP-expressing cells with an excess of LEAP2 or ghrelin fully prevented the F-LEAP2 binding, further confirming that F-LEAP2 specifically binds to GHSR. Fluorescence intensity in the plasma membrane for these conditions was as follows: without fluorescent ligands=0±353 AU, F-LEAP2=15134±3764 AU, F-ghrelin=1531±311 AU, ghrelin plus F-LEAP2=2071±670 AU and LEAP2 plus F-LEAP2=229±370 AU (p<0.0001 for all the conditions compared to F-LEAP2). Thus, F-LEAP2 retained high specificity to GHSR despite the attached fluorescent moiety. Interestingly, it has been shown that the substance P analogue, which was the first reported GHSR inverse agonist [START_REF] Holst | High constitutive signaling of the ghrelin receptor--identification of a potent inverse agonist[END_REF], also fails to induce internalization of GHSR [START_REF] Holliday | Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor[END_REF]. Thus, LEAP2 not only blocks ghrelin-evoked activity and reduces constitutive GHSR activity but also reduces internalization.

3.5.

In vivo imaging studies. In order to test the capability of F-LEAP2 to label GHSRexpressing neurons in vivo, mice were ICV-injected with F-LEAP2 or vehicle (In vivo experiment 1). After 15 min, mice were euthanized and perfused, and brains were extracted, frozen, coronally cut and visualized in a fluorescence microscope. The brain areas displaying significant F-LEAP2 labeling are outlined in Table 1 and summarized with some representative images in Figure 4. The abbreviations used in the figures and throughout the text are listed in Table 2. In contrast to vehicle-injected mice, F-LEAP2-injected mice showed labeled cells in a variety of brain regions. The highest number and strongest intensity of F-LEAP2 labeled cells were observed in the hypothalamus, with the ARC as the area with highest amount of F-LEAP2 labeled cells (Fig. 4Aa). A relatively high number of F-LEAP2 labeled cells was found in some specific hypothalamic nuclei, such as the VMH (Fig. 4Ab), VMPO (Fig. 4B), SCh (Fig. 4C), PVH (Fig. 4D) and SuM (Fig. 4E), while a rather moderate number of F-LEAP2 labeled cells was found in hypothalamic nuclei, such as the AHA, DMH, Pe and PMV. F-LEAP2 labeled cells were also found in the DG and the Ammon's horns of the hippocampus (Fig. 4F), in the MS of the septum (Fig. 4G), in PV of the thalamus and in the SFO (Fig. 4H). A relatively high number of F-LEAP2-labeled cells was also found in the LDTg (Fig. 4I), which is located at the junction of the midbrain and pons. In the medulla oblongata, F-LEAP2 labeled cells were observed in all three components of the dorsal vagal complex: the AP, the NTS and the DMV (Fig. 4J). The pattern of labeling observed in F-LEAP2-injected mice in all the above referred brain regions is consistent with previous reports describing the distribution of GHSR mRNA in mice [START_REF] Mani | Neuroanatomical characterization of a growth hormone secretagogue receptor-green fluorescent protein reporter mouse[END_REF][START_REF] Perello | Functional implications of limited leptin receptor and ghrelin receptor coexpression in the brain[END_REF][START_REF] Zigman | Expression of ghrelin receptor mRNA in the rat and the mouse brain[END_REF] as well as with the pattern of labeling observed in mice ICV injected with fluorescent ghrelin [START_REF] Cabral | Analysis of brain nuclei accessible to ghrelin present in the cerebrospinal fluid[END_REF]. Interestingly, little number or inconsistent presence of F-LEAP2 labeled cells were found in some brainstem areas, such as the VTA, SN, DR, DTg, EW, Amb and MCPC, where GHSR mRNA expression has been previously reported [START_REF] Perello | Functional implications of limited leptin receptor and ghrelin receptor coexpression in the brain[END_REF][START_REF] Scott | Hindbrain ghrelin receptor signaling is sufficient to maintain fasting glucose[END_REF][START_REF] Zigman | Expression of ghrelin receptor mRNA in the rat and the mouse brain[END_REF]. Similarly, low labeling has been also observed in these brain areas in mice ICVinjected with fluorescent ghrelin [START_REF] Cabral | Analysis of brain nuclei accessible to ghrelin present in the cerebrospinal fluid[END_REF]. Recently, we showed that ICV-injected fluorescent-ghrelin only accesses to few hundred of microns into the brain parenchyma [START_REF] Uriarte | Evidence Supporting a Role for the Blood-Cerebrospinal Fluid Barrier Transporting Circulating Ghrelin into the Brain[END_REF]. Thus, it seems likely that molecules present in the cerebrospinal fluid cannot easily reach brain areas distantly located from the brain ventricular system [START_REF] Perello | Brain accessibility delineates the central effects of circulating ghrelin[END_REF]. Finally, it is worth mentioning that F-LEAP2 labeling was detected in the cell body and the cellular processes of hypothalamic tanycytes, which line the floor of the third ventricle (Fig 4Ac), and in epithelial cells of the choroid plexus (Fig 4Fc). This observation is interesting because these cells form the blood-cerebrospinal fluid barrier and have been proposed to transport hormones, including ghrelin [START_REF] Cabral | Brain circuits mediating the orexigenic action of peripheral ghrelin: narrow gates for a vast kingdom[END_REF], through these compartments.

In order to assess the specificity of F-LEAP2 labeling in vivo, mice were first ICVinjected with vehicle, full-length LEAP2 or ghrelin and afterwards with F-LEAP2 (In vivo experiment 2). After 15 min, mouse brains were obtained, as described above, and the F-LEAP2 signal was estimated in the ARC. In this case, the ARC of mice pretreated with fulllength LEAP2 or ghrelin showed significantly lower F-LEAP2 labeling, as compared to the ARC of mice pretreated with vehicle (Figure 5). Overall, these data indicated that F-LEAP2 retained specificity to GHSR in mice.

3.6. Bioactivity assessment. Since LEAP2 inhibits ghrelin-induced food intake, the bioactivity of F-LEAP2 was estimated by assessing its ability to impair the orexigenic effects of ghrelin in mice (In vivo experiment 3). ICV-injected LEAP2 or F-LEAP2 did not affect food intake (not shown), while ICV-injected ghrelin potently increases food intake, as it is well established. In mice pretreated with the LEAP2 variants, ICV-injected F-LEAP2 induced ~40% reduction of ghrelin-induced food intake, in a similar fashion to that seen for full-length LEAP2 (Figure 6).

CONCLUSIONS

We designed a fluorescent derivative of LEAP2 based on the N-terminal sequence of the natural peptide, which was confirmed to be the active part of the hormone. The synthesis F-LEAP2 is easier than the synthesis of fluorescent variants of ghrelin as it does not contain any posttranslational modification. Since F-LEAP2 does not contain any hydrophobic group, its solubility and handling are also more convenient than probes based on ghrelin. Moreover, F-LEAP2 is devoid of most non-specific binding effects often associated with ghrelin. F-LEAP2 is fully bioactive and displays high affinity and specificity to GHSR. F-LEAP2 does not induce GHSR internalization. Since F-LEAP2 is inverse agonist of GHSR signaling while fluorescent variants of ghrelin are full agonists, F-LEAP2 displays pharmacological properties different to fluorescent ghrelin. As such, F-LEAP2 extends our palette of pharmacological ligands and represents a tool of major interest in fluorescent-based pharmacological analyses of GHSR, including ligand binding and screening assays, dimerization analyzes or tracking of the diffusion and/or compartmentalization behavior of GHSR at the cell surface. In addition, F-LEAP2 represents a valuable tool to gain insights about the biology of GHSR in in vivo settings.

FIGURE LEGENDS

Figure 1: Experimental design of in vivo studies. The figure summarizes the experimental design used for the different studies in which mice received ICV treatments. Panel A depicts the design of the in vivo experiment 1, in which the capability of F-LEAP2 to label the mouse brain was tested. Panel B depicts the design of the in vivo experiment 2, in which the ability of LEAP2 and ghrelin to block F-LEAP2 labeling in the mouse brain was tested. Panel C depicts the design of the in vivo experiment 3, in which the ability of F-LEAP2 and LEAP2 to impair the orexigenic effects of ghrelin in mice was assessed. µm; G-J, 100 µm. All abbreviations used for brain nuclei are listed in Table 1. Figure 5. F-LEAP2 labeling in the mouse brain is impaired by pretreatment with LEAP2 or ghrelin. First and second columns show representative photomicrographs of F-LEAP2 signal in the ARC of mice that were ICV-injected with vehicle alone or containing F-LEAP2, respectively. Third and fourth columns show representative images of F-LEAP2 signal in the ARC of mice that were ICV-injected with LEAP2 or ghrelin, respectively, and further ICV injected with F-LEAP2. Insets show high magnification images of the area marked in the low magnification image. Arrows point F-LEAP2 labeled cells. Scale bars, 100 µm (low magnification), 10 µm (high magnification). Figure 6. F-LEAP2 blocks ghrelin-induced food intake in a similar fashion as seen for full-length LEAP2. Food intake in mice ICV injected with vehicle alone or containing LEAP2 or F-LEAP2 and then with ghrelin. Data represent the mean±SEM. **, p≤0.01.

Figure 2 .

 2 Figure 2. Structure and pharmacological features of F-LEAP2. Panel A depicts the structure of F-LEAP2. Panel B shows saturation binding experiments for F-LEAP2 on HEK293T cells transiently transfected with SNAP-GHSR. Panel C shows the BODIPY-FL GTPγS binding to the subunit of Gαqβ1γ2 induced by GHSR in the absence of ligand or in the presence of ghrelin, LEAP2(1-14)-NH2 or F-LEAP2. Data represent the mean±SEM. ***, p≤0.001 vs. no ligand condition.

  Figure 4. F-LEAP2 labels cells in brain areas known to express GHSR. Representative microphotographs of coronal sections of different brain areas of mice ICV-injected with F-LEAP2. Insets show high magnification images of the area marked in the low magnification image. Arrowheads mark different cells labeled with F-LEAP2. Small arrows point at labeled tanycyte processes. Scale bars: A, 100 µm; A a-c, 20 µm; B-E, 100 µm; F, 200 µm; F a-c, 20 µm; G-J, 100 µm. All abbreviations used for brain nuclei are listed in Table1.

Qualitative estimates of F-LEAP2 signal were made by considering both signal strength and the number of labeled cells: +++, high density; ++, moderate density; +, low density; +/-, inconsistent visualization.