Marina Casiraghi

Elodie Point

Alexandre Pozza

Karine Moncoq

Jean-Louis Banères

Laurent J Catoire
email: laurent.catoire@ibpc.fr

NMR analysis of GPCR conformational landscapes and dynamics

Keywords: GPCR, NMR, energy landscape β1AR: β1-adrenergic receptor β2AR: β2-adrenergic receptor

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

Introduction

GPCRs are the largest family of cell-surface receptors, with more than 800 members identified in the human genome. They are integral membrane proteins constituted by 7 trans-membrane (TM) spanning helices (Fig. 1) which mediate the signal transduction from outside to inside the cell, translating stimuli from a broad panel of ligands (neurotransmitters, hormones, ions, photons, etc.) into biochemical responses [START_REF] Rosenbaum | The structure and function of G-protein-coupled receptors[END_REF]. Ligands with different efficacies are responsible for the fine-tuning of GPCR signaling, as they can block, activate or modulate the activity of the receptors [START_REF] Deupi | Energy landscapes as a tool to integrate GPCR structure, dynamics, and function[END_REF]. The diversity of GPCR-mediated biological outputs results from the ability of most receptors to activate G protein dependent and independent pathways, as a single GPCR can activate different G protein sub-types or other effectors such as arrestins [START_REF] Luttrell | Refining efficacy: allosterism and bias in G protein-coupled receptor signaling[END_REF]. Due to their prominent role in physiological processes, GPCRs represent the target of around 30% of the drugs currently on the market [START_REF] Rask-Andersen | The druggable genome: evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication[END_REF][START_REF] Santos | A comprehensive map of molecular drug targets[END_REF][START_REF] Hauser | Trends in GPCR drug discovery: new agents, targets and indications[END_REF], addressing a broad range of pathologies including neuropsychiatric, cardiovascular, pulmonary and metabolic disorders, cancer, obesity and AIDS [START_REF] Lagerström | Structural diversity of G protein-coupled receptors and significance for drug discovery[END_REF][START_REF] Bockaert | GPCR interacting proteins (GIPs) in the nervous system: Roles in physiology and pathologies[END_REF][START_REF] Persaud | Islet G-protein coupled receptors: therapeutic potential for diabetes[END_REF][START_REF] Nieto Gutierrez | GPCRs: emerging anti-cancer drug targets[END_REF]. Despite recent major advances in the field, there is still an important lack of knowledge regarding GPCR signaling mechanism in association with their manifold pharmacology. This implies that GPCRs are the object of intense study in order to characterize the relationship between their structure, conformational dynamics and function, with important implications for drug design.

Numerous biological functions are closely related to changes in spatial and temporal locations of atoms inside biomolecules. The GPCR-mediated signal transduction phenomenon through the membrane of eukaryotic cells underlies the existence of a certain conformational plasticity that has yet to be understood. Indeed, the functional versatility of GPCRs cannot be explained by a simple two-state model of activation considering only an active and an inactive state, as these receptors are highly dynamic and can explore a wide range of conformations [START_REF] Manglik | The role of protein dynamics in GPCR function: insights from the β2AR and rhodopsin[END_REF][START_REF] Latorraca | GPCR dynamics: structures in motion[END_REF]. GPCRs are able to modify and adapt their shape in response to ligands with different pharmacological profiles, through a series of structural rearrangements that are crucial for the binding of various downstream intracellular partners [START_REF] Urban | Functional selectivity and classical concepts of quantitative pharmacology[END_REF]. The ensemble of conformations that a receptor can adopt can be defined with the concept of conformational landscape [START_REF] Frauenfelder | The energy landscapes and motions of proteins[END_REF], which describes how highly dynamic proteins like GPCRs can exist in an ensemble of conformations in the presence or absence of ligands. In this energy landscape, the ground states, or low-energy conformations, correspond to the global minimum of energy, an ensemble of conformers separated by small kinetic barriers, that are easily overcome in the range of motions in the picosecond to nanosecond (ps-to-ns) timescales (Fig. 2). While most biophysical techniques are able to sample the highly probable low-energy states, higher energy conformers, also known as excited states, are more difficult to study due to the difference in free energies between the ground and the excited states (Boltzmann law). Moreover, the lifetime of those states can be very short in the case of unfavorable activation energy from the ground to the excited state. Under the assumption of a conformational selection process triggering receptor activation, pre-existing conformations or sub-states around the basal state (tiers 1 and 2 in Fig. 2) may explain why different ligands are able to bind a same GPCR, as each conformation would have a differential propensity to interact with a given ligand compared to the others. The ligand binding event would subsequently lead to the selection and/or selective stabilization of a given conformation, thus causing a population shift in the conformational ensemble that would favor the interaction with a specific intracellular partner and therefore engage a biological response. To get a full picture of the functional implications of these different conformers, we need to fully characterize the conformations that populate the energy landscape and their associated relative probabilities (thermodynamics) and the dynamic barriers separating these different conformations during the activation and inactivation of the receptors (kinetics) [START_REF] Henzler-Wildman | Dynamic personalities of proteins[END_REF][START_REF] Smock | Sending signals dynamically[END_REF].

NMR: a powerful tool to investigate energy landscapes at the atomic scale

NMR is one of the most powerful techniques to investigate the energy landscapes of biomolecules, with a spectrum of different methodologies to address i) conformational ensembles, including hidden or sparsely and transiently populated conformers (i.e. with a relative population below 0.5% and a lifetime of less 1 millisecond), and ii) timescales of motions from ps to s and beyond. To do so, NMR can interrogate a vast number of reporters within the protein, the NMR active nuclei, which are 1 H, 15 N and 13 C in proteins, and also 19 F thanks to the use of chemical modifications. The position of a signal, named chemical shift, represents an atom of the receptor in the NMR spectrum, and is dependent on the micro-environment that this atom experiences within the protein. In proteins, 1 H, 15 N and 13 C nuclei may experience a variety of environments that depend on both local and global protein structure. As a result, depending on the localization of the nucleus, the position of the NMR peak can be very sensitive to changes in protein conformation and to the rates of chemical exchange between the different conformations sampled by the protein (Fig. 3). Thus, it is possible to directly observe several co-existing conformations or variations in the conformational ensemble, if these conformations are populated enough and provided that the inter-converting rates of exchange between the different conformers are slow enough compared to the window of NMR observation (directly related to the value of the static magnetic field) experienced by the nucleus under investigation. In other words, two peaks in a spectrum corresponding to two distinct conformations, like in Fig. 3 (right panel), can be observed if the difference in chemical shifts between these two peaks is much greater than the rate of chemical exchange.

NMR is a very useful tool to study the chemical exchanges towards lowly populated (<5%) and transiently formed conformations (few milliseconds at most). This typically concerns excited-state conformers separated by high kinetic barriers that are usually transient to the traditional biophysical methods [START_REF] Baldwin | NMR spectroscopy brings invisible protein states into focus[END_REF]. A number of NMR approaches have been developed to characterize these invisible states (Palmer et al., 2001;[START_REF] Hansen | Using relaxation dispersion NMR spectroscopy to determine structures of excited, invisible protein states[END_REF][START_REF] Clore | Exploring sparsely populated states of macromolecules by diamagnetic and paramagnetic NMR relaxation[END_REF][START_REF] Fawzi | Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR[END_REF][START_REF] Vallurupalli | Studying "invisible" excited protein states in slow exchange with a major state conformation[END_REF]. These methods are based on the use of the relaxation properties of the spin under study that are sensitive to the chemical exchanges [START_REF] Mittermaier | Observing biological dynamics at atomic resolution using NMR[END_REF]. The use of NMR relaxation experiments allows also to quantitatively determine the kinetic barriers separating the different conformations that occur on different timescales by determining the rates of inter-conversion and the populations involved [START_REF] Palmer | Solution NMR spin relaxation methods for characterizing chemical exchange in high-molecular-weight systems[END_REF]. Importantly, when repeated at equilibrium at various temperatures, these NMR experiments open the possibility to i) determine the enthalpic and entropic contributions to the different chemical exchanges detected in the receptor energy landscape and, ii) measure the forward and backward energies of activation involved in these different processes [START_REF] Kay | NMR studies of protein structure and dynamics[END_REF].

The use of artificial membrane environments

Atomic-scale NMR studies of membrane proteins (MPs), and GPCRs in particular, are performed in vitro with a purified material, in conditions that are far from their native membranes, despite some promising in cellulo studies [START_REF] Selenko | Looking into live cells with in-cell NMR spectroscopy[END_REF][START_REF] Ito | Cellular structural biology[END_REF][START_REF] Renault | Cellular solidstate nuclear magnetic resonance spectroscopy[END_REF][START_REF] Theillet | Structural disorder of monomeric α-synuclein persists in mammalian cells[END_REF]. To date, the most used artificial environments for NMR studies on GPCRs are detergents [START_REF] Tanford | Micelle shape and size[END_REF][START_REF] Grisshammer | Purification of recombinant G-protein-coupled receptors[END_REF], micelles/bicelles [START_REF] Vold | Magnetically oriented phospholipid bilayered micelles for structural studies of polypeptides. Does the ideal bicelle exist?[END_REF][START_REF] Sanders | Bicelles: a model membrane system for all seasons?[END_REF][START_REF] Thompson | GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles[END_REF], amphipols [START_REF] Tribet | Amphipols: polymers that keep membrane proteins soluble in aqueous solutions[END_REF][START_REF] Dahmane | Amphipol-assisted in vitro folding of G protein-coupled receptors[END_REF] and lipid nanometric bilayers, also named nanodiscs or HDL particles [START_REF] Bayburt | Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins[END_REF][START_REF] Whorton | A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein[END_REF] (see e.g. [START_REF] Catoire | Micelles, bicelles, amphipols, nanodiscs, liposomes, or intact cells: the hitchhiker's guide to the study of membrane proteins by NMR. Membrane Proteins Production for Structural Analysis[END_REF] for a more detailed description of artificial membrane environments used for in vitro studies of MPs by NMR).

Usually, the choice of the artificial environment is based on its compatibility with the downstream biophysical application, sometimes at the expense of the protein integrity. To date, most of the NMR studies on GPCRs have been conducted in detergent solutions, because the size of the GPCR embedded in a detergent micelle is smaller compared to other membrane-like systems, considering that the quality of signals in solution-state NMR is very sensitive to the size of the protein or protein complex under study. The size dependence is closely related to the overall correlation time, which is inversely proportional to the rate of rotation of the object on itself, which impacts the nuclear spin relaxation properties. The longer the protein takes to rotate in solution on itself, the shorter the lifetime of the NMR signal is, resulting in broad NMR peaks that can even disappear in the spectral background noise.

Although they represent the easiest alternative, detergents are known to destabilize membrane proteins [START_REF] Popot | Amphipols, nanodiscs, and fluorinated surfactants: three non-conventional approaches to studying membrane proteins in aqueous solutions[END_REF][START_REF] Chipot | Perturbations of native membrane protein structure in alkyl phosphocholine detergents: a critical assessment of NMR and biophysical studies[END_REF], potentially altering their activity and function. Also, detergents undoubtedly affect the dynamics of the proteins under investigation (e.g. [START_REF] Chung | Role of detergents in conformational exchange of a G Protein-coupled receptor[END_REF], possibly compromising the interpretation of the conformational landscape, and, above all, they do not recapitulate the features of a native membrane, which has a prominent role in receptor dynamics and conformational modulation [START_REF] Pucadyil | Role of cholesterol in the function and organization of G-protein coupled receptors[END_REF][START_REF] Mondal | How the dynamic properties and functional mechanisms of GPCRs are modulated by their coupling to the membrane environment[END_REF][START_REF] Casiraghi | Functional modulation of a G protein-coupled receptor conformational landscape in a lipid bilayer[END_REF]. Alternatives exist to avoid the use of detergent solutions, like nanodiscs, that mimic a more membrane-like environment that can still be compatible with the acquisition of highly resolved NMR data of GPCRs [START_REF] Kofuku | Functional dynamics of deuterated β2-adrenergic receptor in lipid bilayers revealed by NMR spectroscopy[END_REF][START_REF] Casiraghi | Functional modulation of a G protein-coupled receptor conformational landscape in a lipid bilayer[END_REF][START_REF] Casiraghi | Illuminating the energy landscape of GPCRs: the key contribution of solution-state NMR associated with Escherichia coli as an expression host[END_REF].

Isotope-labeling schemes for large proteins: deuteration

Coordinated with improvements in NMR methodology and hardware, the capacity of NMR to describe the energy landscape of large proteins or protein complexes in solution is closely related to the isotope enrichment method chosen. As mentioned in the former paragraph, the quality of the NMR signal in solution, which concerns both the sensitivity and the resolution, is predominantly determined by the overall size of the object. As the molecular weight increases, NMR spectra will display a lack of spectral resolution, due to the larger number of signals and the unfavorable relaxation properties of the system (increased relaxation rates caused by the slow overall tumbling rate), at the point that the signal deteriorates and might become impossible to detect. The best strategy to overcome those issues is to express the recombinant protein in deuterated media [START_REF] Gardner | The use of 1 H, 13 C, 15 N multidimensional NMR to study the structure and dynamics of proteins[END_REF]. Deuteration improves resolution and the signal-to-noise by reducing the relaxation rates of the remaining protons and other nuclei under investigation such as 15 N and 13 C [START_REF] Kerfah | Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins[END_REF]. This is due to a fundamental physic constant, the gyromagnetic ratio, which is directly related to the frequency of precession of the spin in an external magnetic field. Deuterons have a gyromagnetic ratio 6.7 times weaker than protons, which considerably reduces the dipolar interactions with the neighboring nuclei (remaining 1 H, 15 N and 13 C). The main consequence is that the transverse relaxation rates of observed nuclei become much slower, substantially improving the quality of the NMR signals.

Importantly, the level of deuteration must be as high as possible in the case of large tumbling objects in solution, because the signal deteriorates dramatically due to the remaining protons around the nuclei under investigation. Perdeuteration occurs when all protons have been replaced, except for those that are aimed to be detected, like exchangeable amide protons along the backbone of the protein or protons that have been specifically incorporated during amino-acid biosynthesis or subsequently added by chemical reactions. The use of deuteration, together with the advent of ad hoc NMR experiments dedicated to the study of large protein complexes, opened the way to address complex biological questions like the study of conformational landscapes (e.g. [START_REF] Ruschak | The proteasome antechamber maintains substrates in an unfolded state[END_REF][START_REF] Velyvis | An economical method for production of 1 H, 13 CH3-threonine for solution NMR studies of large protein complexes: application to the 670 kDa proteasome[END_REF][START_REF] Sekhar | Hsp70 biases the folding pathways of client proteins[END_REF][START_REF] Huang | Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study[END_REF], or dynamics and allostery properties [START_REF] Religa | Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR[END_REF][START_REF] Ruschak | Proteasome allostery as a population shift between interchanging conformers[END_REF][START_REF] Zhuravleva | An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones[END_REF][START_REF] Rosenzweig | Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction[END_REF] of very large protein complexes, with a size that can exceed 1 MDa.

GPCR energy landscapes by NMR

Solution-state NMR has been widely applied to the study of the conformational dynamics of a series of GPCRs mostly obtained from expression in the membrane of insect cells (sf9 cells) and subsequently extracted from the membrane by detergents. GPCRs that have been investigated by NMR following this approach are the β2-adrenergic receptor (β2AR) [START_REF] Bokoch | Ligandspecific regulation of the extracellular surface of a G-protein-coupled receptor[END_REF]Liu et al., 2012a;[START_REF] Kofuku | Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region[END_REF][START_REF] Kim | The role of ligands on the equilibria between functional states of a G Protein-coupled receptor[END_REF][START_REF] Nygaard | The dynamic process of β2adrenergic receptor activation[END_REF][START_REF] Kofuku | Functional dynamics of deuterated β2-adrenergic receptor in lipid bilayers revealed by NMR spectroscopy[END_REF][START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF], μ-opioid receptor (μOR) [START_REF] Okude | Identification of a conformational equilibrium that determines the efficacy and functional selectivity of the μ-opioid receptor[END_REF][START_REF] Sounier | Propagation of conformational changes during μ-opioid receptor activation[END_REF] and β1AR [START_REF] Isogai | Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor[END_REF][START_REF] Solt | Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor[END_REF]. More recently, NMR investigations have been conducted on a few receptors expressed in other organisms, such as the A2AR, expressed in yeast [START_REF] Ye | Activation of the A2A adenosine G-protein coupled receptor by conformational selection[END_REF][START_REF] Clark | Ligand modulation of sidechain dynamics in a wild-type human GPCR[END_REF][START_REF] Ye | Mechanistic insights into allosteric regulation of the A2A adenosine G proteincoupled receptor by physiological cations[END_REF][START_REF] Eddy | Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor[END_REF] and BLT2, expressed in Escherichia coli (E. coli) [START_REF] Casiraghi | Functional modulation of a G protein-coupled receptor conformational landscape in a lipid bilayer[END_REF] (see also [START_REF] Casiraghi | Illuminating the energy landscape of GPCRs: the key contribution of solution-state NMR associated with Escherichia coli as an expression host[END_REF] on the advantages and drawbacks of GPCRs expression in different hosts). The body of data collected in these studies highlights the co-existence of different GPCRs conformations at equilibrium, i.e. inactive, active and intermediate states, whose probability and lifetime are modulated by the presence of ligands and effectors with different properties.

GPCRs expressed in insect cells

β2AR, one of the main target of investigation within the GPCR family, has been the first and most extensively characterized receptor by NMR [START_REF] Bokoch | Ligandspecific regulation of the extracellular surface of a G-protein-coupled receptor[END_REF][START_REF] Liu | Biased signaling pathways in β2-adrenergic receptor characterized by 19 F-NMR[END_REF][START_REF] Kofuku | Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region[END_REF][START_REF] Kim | The role of ligands on the equilibria between functional states of a G Protein-coupled receptor[END_REF][START_REF] Nygaard | The dynamic process of β2adrenergic receptor activation[END_REF][START_REF] Kofuku | Functional dynamics of deuterated β2-adrenergic receptor in lipid bilayers revealed by NMR spectroscopy[END_REF][START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF]. Pioneering NMR conformational studies have been conducted by Bokoch and collaborators following the rearrangements of the extracellular surface of the receptor upon ligand binding [START_REF] Bokoch | Ligandspecific regulation of the extracellular surface of a G-protein-coupled receptor[END_REF]. They studied the conformational changes around a salt bridge between the extracellular loop 2 (ECL2) and ECL3, previously observed in crystal structures. Their NMR probes consisted in two 13 C-labeled methyl groups chemically attached through reductive methylation to the ε-NH2 of lysine side chains. The 13 C-dimethyllysines served as conformational probes in 2D 1 H, 13 C correlation NMR experiments that uncovered the existence of three conformations for the receptor in n-Dodecyl β-D-maltoside (βDDM) detergent solution: one conformation (I) predominantly populated in the unliganded state of the receptor or in the presence of a neutral antagonist, another conformation (II) populated in the presence of an inverse agonist and a third one (III) stabilized in the presence of the agonist. These conformations are associated with different functional behaviors: when conformation (I) is predominant, the receptor is able to couple to Gs, in agreement with the concept of basal activity and the efficacy of the neutral antagonist alprenolol; when population (II) is predominant, carazolol (inverse agonist) prevents the coupling of the receptor to Gs; conformation (III), which is predominant in the presence of a full agonist, is associated with a strong coupling to Gs. Although this investigation of β2AR conformational landscape was limited to the solvent-accessible lysines, this study represents an elegant approach to the characterization of ligand-induced changes in the extracellular surface of the receptor [START_REF] Bokoch | Ligandspecific regulation of the extracellular surface of a G-protein-coupled receptor[END_REF].

A different labeling scheme was exploited by Liu and collaborators for the investigation of the conformational changes of the β2AR in βDDM in response to ligand binding (Liu et al., 2012a). Three cysteines in the cytoplasmic part of the receptor, on trans-membrane helices VI (TMVI) and VII (TMVII), were covalently labeled with 2,2,2 trifluoroethanethiol (TET) [START_REF] Klein-Seetharaman | NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: applicability of solution 19 F NMR[END_REF]. 19 F-NMR data were deconvoluted using a double-Lorentzian function in order to obtain quantitative information about the conformational equilibria. The observation of 19 F-NMR spectra upon addition of ligands with different pharmacological properties revealed the presence of two conformations for each of these two helices, whose relative populations were modulated by various ligands. Hence, the binding of an agonist would favor one of the conformations of helix VI that would correspond to the G protein specific active state, while β-arrestinbiased ligands would populate the conformational state of helix VII that would favor the interaction with βarrestin (Liu et al., 2012a).

19 F-NMR has been successfully used in other studies on the β2AR. One study introduced the use of a novel detergent, the maltose neopentyl glycol-3 (MNG-3), which displays a very low critical micellar concentration [START_REF] Chae | Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins[END_REF]. 19 F-NMR investigations on chemically modified Cys265 at the intracellular extremity of TMVI of β2AR revealed the presence of different conformations: two distinct inactive states, an intermediate state en route to activation, and a predominant active state in the presence of the G protein mimic [START_REF] Kim | The role of ligands on the equilibria between functional states of a G Protein-coupled receptor[END_REF]. The observation of four states instead of the previous two was possible thanks to the use of this new generation of detergents, which compared to βDDM display a slower off rate, resulting in a better spectral resolution [START_REF] Chung | Role of detergents in conformational exchange of a G Protein-coupled receptor[END_REF]. Experiments conducted with the unliganded receptor also highlighted the coexistence of three populations, two predominant one and a minor one [START_REF] Kim | The role of ligands on the equilibria between functional states of a G Protein-coupled receptor[END_REF].

Those findings were further completed by another study, published by Manglik and collaborators in 2015 [START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF]. This time, β2AR reconstituted in MNG micelles was studied by 19 F-NMR in combination with double electron-electron resonance (DEER) experiments. Cys265 at TMVI was labeled with 13 C 19 F3-3-bromo-1,1,1-trifluoroacetone ( 13 C 19 F3-BTFA) for NMR investigations, while 3-(2-Iodoacetamido)-Proxyl (IA-Proxyl) was used to label TMIV and TMVI of the β2AR for DEER studies. They confirmed the coexistence of two inactive states for the unliganded receptor, that interconverted in the ms regime. The addition of an agonist shifted the equilibrium towards the active state but in an incompletely manner without the presence of the G protein, resulting in the co-existence of different conformational states. Complete transition to the active state was only observed in the presence of the G protein or a nanobody, a G protein mimic. The two inactive conformations in the unliganded state seemed to correspond to the intact and broken "ionic lock" [START_REF] Nygaard | Ligand binding and micro-switches in 7TM receptor structures[END_REF], which interconvert due to the low energy barriers between the two populations. They hypothesized that the break of the ionic lock could represent an important step towards activation, a mechanism that might be conserved among other receptors of the family [START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF].

13 C-labeled methyl groups of methionines were used to detect the presence of two inactive states at equilibrium, and a third active one in the presence of the agonist in the β2AR [START_REF] Kofuku | Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region[END_REF]. Interestingly, with the addition of a partial agonist, the receptor was in equilibrium between inactive and active conformations. In the presence of an inverse agonist they observed a minor active conformation in equilibrium with the inactive one, corresponding to the presence of a residual basal activity.

Nygaard and collaborators used 13 CH3 methionines to gain access to the TM part of the receptor, not accessible by 19 F labeling, to study β2AR conformational changes [START_REF] Nygaard | The dynamic process of β2adrenergic receptor activation[END_REF]. They specifically followed Met82, located below the orthosteric pocket in TMII, and Met215 and Met279, located halfway between the ligand binding pocket and the cytoplasmic side of TMV and TMVI [START_REF] Nygaard | The dynamic process of β2adrenergic receptor activation[END_REF]. In the unliganded state or in the presence of an inverse-agonist (corazalol), the receptor showed the co-existence of two peaks in the ms timescale, that according to molecular dynamics simulations seemed to correspond to two distinct inactive conformations. They could also observe that agonist alone (BI-167107) did not stabilize the fully active conformation, that became the most populated one only in the presence of the G protein mimetic, the nanobody Nb80, as confirmed by other studies of the group [START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF].

The studies mentioned so far represent milestones in the description of GPCR conformational landscape. However, they are characterized by some caveats: i) as mentioned in § 4, the protonated environment, which precludes the detection of subtle but important variations in the conformational landscape, and ii) the lack of a native environment, as all the studies mentioned above were conducted in detergents. The choice of detergents is mostly related to the size of the object under study, which needs to be as small as possible taking into account the impossibility to perdeuterate the receptor. Unfortunately, detergents undoubtedly affect the dynamics of the protein under investigation compared to a lipid environment [START_REF] Chung | Role of detergents in conformational exchange of a G Protein-coupled receptor[END_REF].

To face those issues, the group of Shimada published a study with two major breakthroughs: the use of lipid nanodiscs [START_REF] Bayburt | Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins[END_REF][START_REF] Ritchie | Reconstitution of membrane proteins in phospholipid bilayer nanodiscs[END_REF] and a partial deuteration of the β2AR receptor in insect cells [START_REF] Kofuku | Functional dynamics of deuterated β2-adrenergic receptor in lipid bilayers revealed by NMR spectroscopy[END_REF]. Partial deuteration was made possible through the addition of 14 types of deuterated amino acids whose choice was based on the 3D crystal structure of the receptor to identify the most common residues around the 13 CH3 groups of methionines. Only selective amino acid deuteration could be achieved, as the growth of insect cell is highly affected in D2O solutions. The amino acid deficient medium was then supplemented with 13 C-methionines. Upon partial deuteration, spectra sensitivity was increased by more than 5 folds. Compared to a similar previous study of the group, in the presence of a partial agonist two signals were detected in nanodiscs, while only one was observed previously in βDDM micelles, suggesting lower exchange rates between inactive and active conformations in nanodiscs compared to βDDM. Similarly, the active population detected in nanodiscs was higher compared to the one observed in a detergent solution [START_REF] Kofuku | Functional dynamics of deuterated β2-adrenergic receptor in lipid bilayers revealed by NMR spectroscopy[END_REF].

A similar approach was used to investigate another receptor of the class A family, the μOR [START_REF] Okude | Identification of a conformational equilibrium that determines the efficacy and functional selectivity of the μ-opioid receptor[END_REF]. In this case, 8 amino acids were deuterated, and the 2D 1 H, 13 C correlation experiments were conducted by following 7 methyl-labeled methionines of the receptor in a detergent solution. The authors suggested an equilibrium between an open conformation, stabilized by the full agonist (DAMGO), and a closed one in the presence of the antagonist (naloxone). When the agonist is present, multiple open conformations coexisted at equilibrium, linked to both the G-protein and β-arrestin signaling pathways. In the presence of the agonist morphine, the receptor was in equilibrium between the closed and open conformations. Biased ligands were able to further switch the equilibrium towards one or the other pathway according to their pharmacology [START_REF] Okude | Identification of a conformational equilibrium that determines the efficacy and functional selectivity of the μ-opioid receptor[END_REF].

The μOR in a detergent solution was also investigated with the use of dymethylated lysines [START_REF] Sounier | Propagation of conformational changes during μ-opioid receptor activation[END_REF]. Nine dimethylated extracellular and intracellular lysines were studied with 2D 1 H, 13 C correlation experiments which confirmed that the binding of the high-affinity agonist, BU72, was not sufficient to fully stabilize the active conformation at the intracellular extremities of TMV and TMVI, which required G protein stabilization (or the nanobody in this case), in agreement with previous studies [START_REF] Nygaard | The dynamic process of β2adrenergic receptor activation[END_REF][START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF]. The study also underlined the weak allosteric coupling between the extracellular μOR binding domain and the G-protein coupling interface, as observed for the β2AR [START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF]. They also investigated structural modifications at the intracellular loop 1 (ICL1) and TMVIII upon addition of the BU72 ligand, concluding that the allosteric coupling between the ligand binding pocket and the ICL1/TMVIII seems to be stronger compared to TMV/TMVI [START_REF] Sounier | Propagation of conformational changes during μ-opioid receptor activation[END_REF]. This effect was not ligand-dependent, as observed with other ligands. They hypothesized that ICL1 and TMVIII are probably the first domains to engage in G protein binding, followed by stabilization of the TM core. This study nicely completed the X-ray structure obtained for this receptor in the presence of the agonist BU72 [START_REF] Huang | Structural insights into μ-opioid receptor activation[END_REF].

Another approach that has been implemented for the NMR studies of GPCRs is the use of thermostabilizing mutations, a strategy that was initially developed to improve the quality of crystals without necessarily the need to add sub-domains from other proteins to increase the crystalline contacts [START_REF] Serrano-Vega | Conformational thermostabilization of the β1adrenergic receptor in a detergent-resistant form[END_REF][START_REF] Lebon | Agonistbound adenosine A2A receptor structures reveal common features of GPCR activation[END_REF][START_REF] Magnani | A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies[END_REF]. A first study conducted in detergent investigated the 28 15 N-labeled valines distributed in the β1AR in a detergent solution to get insights into ligand binding and receptor activation [START_REF] Isogai | Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor[END_REF]. They observed that agonist binding, even in the absence of a G protein mimic, had a heterogeneous outcome at the extracellular side, while it induced initial changes in the conformational equilibrium of TMV, favoring the conformation observed in the G protein complex of β2AR [START_REF] Rasmussen | Structure of a nanobody-stabilized active state of the β2-adrenoceptor[END_REF]. They also reported that the stabilization of GPCR fully active conformation required the binding of an agonist and an intracellular partner, as already highlighted in other studies [START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF][START_REF] Sounier | Propagation of conformational changes during μ-opioid receptor activation[END_REF]. However, to observe a complete shift towards the active state in the presence of the nanobody, two thermostabilizing mutations had to be reverted (Y227 5.58 and Y343 7.53 ), significantly reducing the thermal stability of the receptor [START_REF] Isogai | Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor[END_REF].

Another recent study on a thermostabilized β1AR in detergent investigated, through 13 C-labeled methionines, the role of different ligands and G-protein mimic nanobodies in GPCR activation [START_REF] Solt | Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor[END_REF]. The study suggested an equilibrium between an inactive (I) and a pre-active state (A), the latter being more populated in the presence of a full agonist. This pre-active state was believed to sample dynamics in the order of the μs-ms timescale, that were associated to large amplitude motions at both the extracellular and intracellular side of the receptor. In contrast to observations with the β2AR [START_REF] Nygaard | The dynamic process of β2adrenergic receptor activation[END_REF][START_REF] Kim | The role of ligands on the equilibria between functional states of a G Protein-coupled receptor[END_REF][START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF], the unliganded receptor or the receptor bound to low-affinity ligands displayed more restricted dynamics compared to the full agonist-bound receptor. According to these observations, agonist-bound receptor appeared to be more dynamic than the agonist-receptor-nanobody complex or the receptor coupled to the nanobody only. They interpreted the data by hypothesizing that the highly dynamic form of the agonist-bound receptor is able to sample different active-like conformational states that are primed to bind the different intracellular signaling effectors [START_REF] Solt | Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound β1-adrenergic receptor[END_REF]. To be noticed, as the analysis of the dynamic of the receptor was based on variations in peak intensities only, it would be interesting to incorporate spin relaxation properties to confirm this interpretation.

GPCRs expressed in yeast (Pichia pastoris) or E. coli

All the aforementioned studies were based on the expression of GPCRs in the membrane of insect cells (sf9 cells) followed by the extraction of the receptor from the membrane with the help of detergents. Despite the tremendous amount of information obtained, this methodology remains limited by the impossibility of perdeuterating the receptor, which hampers the detection of some conformations and subtle changes in the conformational landscape that might have important functional implications in the context of receptor activation and signaling. Also, i) chemical modifications of amino acids within the receptors might alter the dynamics of the system under study to an extend that is difficult to be judged, even when the coupling to ligands or G protein is still retained; ii) thermostabilization usually reduces or modifies the dynamics of the system; iii) except for one case [START_REF] Kofuku | Functional dynamics of deuterated β2-adrenergic receptor in lipid bilayers revealed by NMR spectroscopy[END_REF], all the above-mentioned studies were conducted in detergent solutions, that are far from reproducing a membrane-like environment that undoubtedly impact membrane protein dynamics and conformational modulation [START_REF] Pucadyil | Role of cholesterol in the function and organization of G-protein coupled receptors[END_REF][START_REF] Mondal | How the dynamic properties and functional mechanisms of GPCRs are modulated by their coupling to the membrane environment[END_REF][START_REF] Chipot | Perturbations of native membrane protein structure in alkyl phosphocholine detergents: a critical assessment of NMR and biophysical studies[END_REF]. To overcome those limitations, in the recent years, other expression systems for GPCRs production have come to the spotlight, in particular yeast and E. coli, whose main advantage is the capacity to grow in very hostile conditions, typically 100%-D2O media. Thanks to the use of appropriate labeled precursors associated with the expression a growth in 100%-D2O media, high-resolution NMR has been shown to be accessible to large systems, up to 1MDa [START_REF] Kay | NMR studies of protein structure and dynamics[END_REF][START_REF] Religa | Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR[END_REF][START_REF] Ruschak | The proteasome antechamber maintains substrates in an unfolded state[END_REF][START_REF] Kay | New views of functionally dynamic proteins by solution NMR spectroscopy[END_REF].

Yeast has often been used for expression of membrane proteins due its inexpensiveness, ease of use, good scalability and genetic flexibility. Yeast has grown particular interest for NMR studies on GPCRs because it is a eukaryotic system and it is able to grow in a deuterated environment [START_REF] Clark | On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies[END_REF]. However, achieving a good protein expression yield is not always straightforward, particularly in D2O media. This is an important factor if considering that NMR is a technique that requires a lot of material (i.e. few tens of μM to look at conformational ensembles to few hundreds of μM to investigate kinetic barriers, for a sample volume of 150-300 μL). Usually the combination of multiple strategies is required to get a good yield of expressed protein in yeast, and to date only a few GPCRs have successfully been expressed in mg amount in P. pastoris, among which the A2A adrenoceptor [START_REF] O'malley | High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2A receptor[END_REF][START_REF] Hino | Gprotein-coupled receptor inactivation by an allosteric inverse-agonist antibody[END_REF] and the histamine H1 receptor [START_REF] Hino | Gprotein-coupled receptor inactivation by an allosteric inverse-agonist antibody[END_REF]. The first NMR study published on the A2AR consisted in the trifluoromethyl labeling of a modified V229C residue located at the cytoplasmic end of TMVI [START_REF] Ye | Activation of the A2A adenosine G-protein coupled receptor by conformational selection[END_REF]. 19 F-NMR experiments revealed the presence of two inactive and two active states for the unliganded receptor, in contrast to what observed in the case of the β2AR [START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF]. In this "loosely coupled" ensemble, the equilibrium was shifted towards the two active states upon addition of partial or full agonists.

The exchange observed between the two inactive states was in the ms regime, while between the inactive and active states the exchange occurred at a slower rate, in the second timescale, probably due to higher activation barriers. Remarkably different from the case of the β2AR, which is biased towards the inactive state unless the concomitant addition of the agonist and nanobody [START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF], 70% of the unliganded A2AR was already in the active states. The authors suggested that probably the active states associated with the unliganded β2AR are very weakly or shortly populated, and therefore extremely exchange-broadened to the point that they cannot be easily detected.

A recent study from the same group used 19 F-NMR to study the effect of cations on the functional states of A2AR in a detergent solution (MNG) [START_REF] Ye | Mechanistic insights into allosteric regulation of the A2A adenosine G proteincoupled receptor by physiological cations[END_REF]. The data collected confirmed the findings published in the previous article, with a conformational landscape consisting in an ensemble of inactive, intermediate and active states modulated by the addition of ligands. Sodium was reported to have a negative allosteric effect on the activation of GPCRs [START_REF] Christopoulos | G protein-coupled receptor allosterism and complexing[END_REF][START_REF] Liu | Structural basis for allosteric regulation of GPCRs by sodium ions[END_REF][START_REF] Fenalti | Molecular control of δ-opioid receptor signaling[END_REF], as it seemed to favor the inactive and intermediate states at the expense of the active one, an effect that seemed to be concentration-dependent. 23 Na NMR measurements revealed that the binding of sodium was transient, characterized by a residency time of 480 μs on the receptor. They also hypothesized that the release of sodium from the binding site in the unliganded receptor might contribute to the activation process. In the same study the role of other cations, like calcium and magnesium, was also investigated by MD simulations, which suggested a positive allosteric role for the cations through their binding to the acidic residues located in ECL2 and ECL3, described as long-lived electrostatic interactions. Cation binding to these residues would also bring TMV and TMVI closer, facilitating TMVI intracellular rotation [START_REF] Ye | Mechanistic insights into allosteric regulation of the A2A adenosine G proteincoupled receptor by physiological cations[END_REF].

The role of sodium was also addressed with another approach, by looking at the protonated and 13 Clabeled methyl groups located at the extremity of the side-chain of isoleucines of the A2AR in a detergent solution. The receptor was produced in 100%-D2O leading to an almost perdeuterated receptor (~90%) [START_REF] Clark | Ligand modulation of sidechain dynamics in a wild-type human GPCR[END_REF]. The improved deuteration allowed the acquisition of 2D 1 H, 13 C NMR experiments, even though the incorporation efficiency of the isotopic labeled probes was estimated to be only around 50%. The 2D 1 H, 13 C correlation spectra were recorded in the presence of the agonist NECA or the inverse agonist ZM241385. 20 peaks out of 29 of the δ1-CH3 of isoleucines labeled were assigned and the ligand-binding changes observed by NMR were analyzed in the context of previous structural data. A too high sodium concentration seemed to suppress the large structural changes that occurred to the A2AR receptor when bound to ligands, and the study of the side-chains motions of several regions of the receptor showed different conformational patterns depending on whether the receptor was bound to an agonist or an inverse agonist [START_REF] Clark | Ligand modulation of sidechain dynamics in a wild-type human GPCR[END_REF].

The A2AR was again investigated in a detergent solution in a partially deuterated environment [START_REF] Eddy | Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor[END_REF]. 15 N uniformly labeled A2AR was used to investigate the role of Asp52 2.50 as a part of an allosteric hinge between the orthosteric binding pocket and the intracellular side of the receptor. NMR signals from residues located at both the extracellular and at the intracellular sides of the receptor were recorded both for the A2AR and its version with the allosteric switch off, A2AR[D52N] [START_REF] Massink | Sodium ion binding pocket mutations and adenosine A2A receptor function[END_REF]. To confirm that the A2AR from P. pastoris had the same properties of the receptor expressed in insect cells, ligandbinding experiments were performed together with the crystallization of the receptor in the presence of an antagonist. Among the tryptophan and glycine residues that they could assign by NMR, they followed Trp246 6.48 which belongs to the "toggle switch" at the bottom of the ligand binding pocket on TMVI [START_REF] Rosenbaum | The structure and function of G-protein-coupled receptors[END_REF][START_REF] Nygaard | Ligand binding and micro-switches in 7TM receptor structures[END_REF]. They suggested a coupling between the toggle switch and Asp52 2.50 , based on the loss of the signaling-related conformational dynamics at the intracellular surface for the A2AR[D52N], which correlated with changes in the local environment explored by the Trp246 6.48 , in association with the activation of the receptor [START_REF] Eddy | Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor[END_REF].

E. coli has also been employed to conduct NMR studies on GPCRs. This host is closely related to the most robust and cost-effective strategy to study large proteins or protein complexes by high-resolution solution-state NMR [START_REF] Goto | A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15 N, 13 C, 2 H-labeled proteins[END_REF][START_REF] Tugarinov | Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins[END_REF][START_REF] Tugarinov | Methyl groups as probes of structure and dynamics in NMR studies of high molecular weight proteins[END_REF][START_REF] Tugarinov | Isotope labeling strategies for the study of high molecular weight proteins by solution NMR spectroscopy[END_REF][START_REF] Kay | Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view[END_REF]. This bacterial host displays a series of advantages: it is an established system for heterologous membrane proteins production, inexpensive, easy to genetically manipulate, and above all, it is capable of expressing MPs at a high yield in 100%-D2O media (for GPCRs, typically 0.5 to 5 mg per liter of culture of purified protein). The main disadvantages are the lack of post-translational modifications and the fact that high levels of expression may result in inclusion bodies formation, for which an efficient folding strategy is needed (for more details, see [START_REF] Casiraghi | Illuminating the energy landscape of GPCRs: the key contribution of solution-state NMR associated with Escherichia coli as an expression host[END_REF].

To maximize production within E. coli, GPCRs can be expressed as inclusion bodies (Bane ̀res et al., 2003;[START_REF] Arcemisbehere | Leukotriene BLT2 receptor monomers activate the Gi2 GTP-binding protein more efficiently than dimers[END_REF][START_REF] Bane ̀res | New advances in production and functional folding of G-proteincoupled receptors[END_REF]. Indeed, folding of membrane proteins from inclusion bodies no longer represents a bottleneck [START_REF] Popot | Folding membrane proteins in vitro: a table and some comments[END_REF] and this includes also the GPCR family [START_REF] Dahmane | Amphipol-assisted in vitro folding of G protein-coupled receptors[END_REF]. Such strategy allowed to study the conformational ensemble of the low affinity leukotriene receptor BLT2 [START_REF] Casiraghi | Functional modulation of a G protein-coupled receptor conformational landscape in a lipid bilayer[END_REF]. BLT2 was expressed in E. coli in 100% D2O in the presence of labeled precursors which led to the 13 C-labeling of protonated methyl groups of methionines and isoleucines (δ1 position). This concerned five methionines (including the two TM residues Met105 3.36 and Met197 5.54 ) and one single isoleucine (Ile229 6.40 , on the intracellular side of TMVI) [START_REF] Casiraghi | Functional modulation of a G protein-coupled receptor conformational landscape in a lipid bilayer[END_REF]. The receptor was reconstituted into lipid nanodiscs and underwent a full panel of characterization assays to demonstrate that BLT2 from E. coli was fully active and stable prior to NMR. Ligand binding data and G protein activation experiments confirmed that the receptor recapitulated the functional characteristics of a GPCR expressed in eukaryotic cells. The high-quality NMR data recorded allowed to monitor the subtle changes in BLT2 conformational ensemble in response to different ligands. For the unliganded receptor, different co-existing conformations at equilibrium were detected: two inactive states and an intermediate one, in agreement with previous studies [START_REF] Kim | The role of ligands on the equilibria between functional states of a G Protein-coupled receptor[END_REF][START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF] and an active or active-like conformation. This activelike state observed for the unliganded receptor was never observed before and probably corresponds to a low-populated conformer that become visible thanks to the improved resolution of this method. The addition of an agonist, in parallel with measurements of GTP binding to the G protein, confirmed that this population corresponded to the active state [START_REF] Casiraghi | Functional modulation of a G protein-coupled receptor conformational landscape in a lipid bilayer[END_REF]. The use of nanodiscs allowed the investigation at the atomic scale of the role of the lipid bilayer in GPCR modulation [START_REF] Pucadyil | Role of cholesterol in the function and organization of G-protein coupled receptors[END_REF]. In particular, they confirmed that negatively charged cholesteryl hemisuccinate, an analogue of cholesterol used for GPCR structural studies [START_REF] Kuszak | Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2[END_REF], has a positive allosteric effect on BLT2 activity. This study proved the importance of receptor deuteration, that otherwise would have hindered the observation of several co-existing sub-states with possible important functional implications. Perdeuteration also allows to work with nanodiscs, one of the best membrane-like systems for the in vitro study of the membrane impact on GPCRs function.

Concluding remarks

During the past decade, NMR proved to be a powerful technique to investigate the energy landscape of GPCRs. The pivotal studies aforementioned have highlighted some fundamental features that occur during the activation of these proteins. NMR investigations supported the evidence that agonist binding and the consequent receptor activation is a multi-step process, that involves different conformational states. Depending on the level of resolution achieved by NMR several conformations have been detected, corresponding to inactive states, intermediates towards activation, and active or active-like states. The probability and duration of those states depends on the presence of ligands with different pharmacology properties, intracellular effectors and allosteric modulators like ions or lipids. NMR is a very efficient tool to characterize the shift in the populations equilibrium and the relative stability between conformations, as the rearrangements that occur at a structural level on the receptor are detected as chemical shift variations at physiological temperatures. Investigations on GPCR dynamic landscape have also confirmed important insights into receptor function, as the stabilization of a given conformation seems to preferentially engage some signaling pathways rather than others. While GPCR energy landscape has been widely studied for several receptors, the next step in the characterization of GPCR dynamics by NMR consists in the determination of the kinetic barriers that separate the different conformational sub-states. This will help to shed light on the transitions between the distinct states that populate the conformational landscape and will possibly detect the presence of additional sub-states not observed yet because of too lowly populated or transiently formed. To do so, NMR relaxation experiments have to be conducted in order to assess if a chemical exchange exists between the observed conformations or towards lowly and transient states [START_REF] Sekhar | NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers[END_REF]. The characterization of kinetic barriers within these complex allosteric proteins will provide important information regarding activation energies separating the different sub-states and their modulation by ligands. Above fundamental issues, this should greatly help the design of new drugs to block or activate these receptors.

Captions

Fig. 1. Topology of GPCRs. (A) GPCRs are integral membrane proteins constituted by seven transmembrane helices (in orange), three extracellular loops (ECLs, in green) and three intra-cellular loops (ICLs, in purple). In the case of class B, C or F GPCRs, the N-terminus presents a structured extra-cellular domain (schematically represented by a dotted circled). (B,C,D) Cartoon representations of the crystal structure of leukotriene B4 BLT1 receptor (PDB ID: 5X33; [START_REF] Hori | Na+-mimicking ligands stabilize the inactive state of leukotriene B4 receptor BLT1[END_REF] parallel to the membrane (B), from the extracellular side (C) and from the cytoplasmic side (D). α-helices and β-sheet secondary structure elements are represented with cylinders and arrows, respectively.

Fig. 2. Energy landscape.

Illustration with one-dimensional cross-section through the hypersurface energy landscape of a biomolecule. Formally, an energy landscape describes all the possible conformations adopted by a biomolecule and the energy levels associated. The free energy of Gibbs, which corresponds to a thermodynamic potential, is traditionally used in such diagrams to describe chemical exchanges. This diagram describes a chemical exchange between two main populations, pA and pB, where each state contains several sub-states separated by low energy barriers. Populations A and B follow a Boltzmann distribution based on their difference in their free energies, indicated by ΔGAB. The barriers between these states (ΔG ‡ (kA→B) and ΔG ‡ (kB→A)) determine the rates of interconversion (kA→B and kB→A), which are directly related to the forward and backward energies of activation. Importantly, the energy landscape defines the amplitude and timescale of protein motions in direct relation with the energy barriers than can be classified in three tiers, following the definition introduced by Frauenfelder and collaborators [START_REF] Ansari | Protein states and proteinquakes[END_REF]. Tiers 2 and 1 concern fast motions between a large number of structurally similar sub-states, in the ps and ns timescales respectively. In these tiers, the kinetic barriers are low, which also means that the probabilities of transition between the different sub-states are quite high. Conversely, in Tier 0, the probability of transition is much lower and concerns slower motions, in the μs and ms timescales, leading to a conformation B that can be quite different than conformation A because it concerns a priori collective motions of atoms. In the case of GPCRs, the binding of a ligand could modify the energy landscape as illustrated here (dark to light blue curve), by stabilizing the excited state B and increasing its population. According to the energy landscape formalism, GPCRs that display a substantial basal activity will display more favorable energy of activation toward state B compared to GPCRs that display a low or a lack of basal activity. The subsequent binding of a G protein could also stabilize the receptor by decreasing its Gibbs free energy, as showed in various NMR studies (e.g. [START_REF] Manglik | Structural insights into the dynamic process of β2-adrenergic receptor signaling[END_REF]. Hence, to fully understand how GPCRs function, in complement to atomic structures of the different sub-states, the relative probabilities between these different sub-states (thermodynamics) and the different dynamic barriers (kinetics) separating these sub-states have to be characterized (figure adapted from Henzler-Wildma and Kern, 2007; reprinted by permission from Springer Nature). 13 C or 1 H, 15 N correlation experiments. In the simulated 2D 1 H, 13 C experiment represented here, the simulated peaks indicated with various colors correspond to one or more protons (i.e. chemically equivalent like the three protons in a methyl group for instance) attached to a same 13 C. (Left) When the exchange rate is fast compared to the chemical shift difference between conformations A and B, the position of the observed peaks is directly proportional to populations A and B. At the limit of very fast exchange on the NMR chemical shift timescale, peaks have the same shape. In the intermediate exchange regime, i.e. when the exchange rate becomes similar to the chemical shift difference, the signals broaden and shift at the same time. (Right) In the condition of a slow exchange, with populations A and B decreasing and increasing, peak A (blue) will for instance decrease in intensity as peak B (red) increases, with no change in chemical shifts occurring for both peaks. When peaks A and B do not display the same shape, it becomes necessary to take into account the relaxation properties of both states in order to accurately determined the populations involved in the chemical exchange (figure adapted from [START_REF] Williamson | Using chemical shift perturbation to characterize ligand binding[END_REF] reprinted by permission from Elsevier).

Fig. 3 .

 3 Fig. 3. The shape and chemical shift of two-dimensional NMR peaks depend on the exchange rate between two conformations A and B. The chemical shift is very sensitive to structural changes in a biomolecule and can be measured with high accuracy by NMR. Most of the NMR studies of conformational ensembles of GPCRs are based on 2D 1 H,13 C or 1 H, 15 N correlation experiments. In the simulated 2D 1 H, 13 C experiment represented here, the simulated peaks indicated with various colors correspond to one or more protons (i.e. chemically equivalent like the three protons in a methyl group for instance) attached to a same

  Fig. 1 875

Acknowledgments

This work was supported by grants from the Centre National de la Recherche Scientifique, Universités de Montpellier and Paris Diderot, Agence Nationale de la Recherche (ANR-17-CE11-0011), Laboratoire d'Excellence (LabEx) DYNAMO and Equipements d'Excellence (EQUIPEX) CACSICE from the French Ministry of Research, and a postdoctoral fellowship Marie Curie Global Fellowship for M.C. ("AlloGPCR"-799376). The authors acknowledge Soumaya Zlitni for the diligent proofreading of this manuscript.


Sylvie Picard

Hélène Hanaire

Yves Reznik

Pierre-Yves Benhamou

Salha Fendri

Lise Dufaitre

Eric Leutenegger

Bruno Guerci

E Bonnemaison-Gilbert

P Barat

Pascal Pr
email: pascal.barat@chu-bordeaux.fr

Barat

Optimization of Insulin Regimen and Glucose Outcomes with Short-Term Real-Time Continuous Glucose Monitoring in Adult Type 1 Diabetes Patients with Suboptimal Control on Multiple Daily Injections: The Adult DIACCOR Study

Keywords: type 1 diabetes, continuous glucose monitoring (CGM), multiple daily injections (MDI), continuous subcutaneous insulin infusion (CSII), pediatrics, hypogl 1. Introduction

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

Most of the cases of type 1 diabetes (T1D) are diagnosed in children and adolescents. T1D management in children has unique characteristics, as food intake and physical activity are quite unpredictable in toddlers while hormonal changes in growing children may alter insulin sensitivity and diabetes management. Although insulin analogs, insulin pumps, and insulin bolus calculators have been helpful with these issues, optimal glucose control often remains very difficult to achieve in youths. Nevertheless, chronic hyperglycemia can ultimately lead to micro-and macrovascular complications [START_REF] Group | Modern-day clinical course of type 1 diabetes mellitus after 30 years' duration: the diabetes control and complications trial/epidemiology of diabetes interventions and complications and Pittsburgh epidemiology of diabetes complications experience (1983-2005)[END_REF] as well as to cognitive impairment [START_REF] Nunley | Clinically Relevant Cognitive Impairment in Middle-Aged Adults With Childhood-Onset Type 1 Diabetes[END_REF] and there is also concern about the long-term consequences of hypoglycemia and mostly severe hypoglycemia (SH). In fact, when SH events occur before the age of 6 years, subtle changes in cognitive performances can be observed at young adult age [START_REF] Ly | Neurocognitive outcomes in young adults with early-onset type 1 diabetes: a prospective follow-up study[END_REF].

Adequate glucose control can be reached in children with T1D with either multiple daily insulin injections (MDI) or continuous subcutaneous insulin infusion (CSII), which usually leads to better glucose control, as was reported in the SWEET registry (16,570 children worldwide) [START_REF] Szypowska | Insulin pump therapy in children with type 1 diabetes: analysis of data from the SWEET registry[END_REF] in which 44.4% of the children were treated with CSII. In France, CSII cost is entirely covered by the national health insurance. However, some patients or their family remain reluctant to use CSII treatment. Pump rejection has been reported in young patients for mainly social/psychological reasons, while pump discontinuation was associated with previous poor glucose control [START_REF] Wong | T1D Exchange Clinic Network. Evaluation of Pump Discontinuation and Associated Factors in the T1D Exchange Clinic Registry[END_REF].

It was clearly shown in adults that the use of long-term real-time continuous glucose monitoring (RT-CGM) helps to identify glucose profiles and results in HbA1c lowering, improvement in glucose variability, and reduction in the incidence of hypoglycemia events. Unlike in adults, early trials using CGM, such as the JDRF study, failed to show a benefit of RT-CGM in children or teens [START_REF] Tamborlane | Continuous glucose monitoring and intensive treatment of type 1 diabetes[END_REF]. However, only 30% (in the 15-24-year-old group) to 50% (in the 8-14-year-old group) of the young patients from the JDRF study used the sensor more than 6 days per week, whereas it was shown that CGM efficacy is clearly related to adherence [START_REF] Raccah | Incremental value of continuous glucose monitoring when starting pump therapy in patients with poorly controlled type 1 diabetes: the RealTrend study[END_REF]. This points out the difficulty of wearing a glucose sensor on a long-term basis in youths even with the newest devices: in the T1D Exchange Registry, 26% of the 18-25-year-olds were CGM users compared with 40-48% in the older age groups [START_REF] Tanenbaum | Diabetes Device Use in Adults With Type 1 Diabetes: Barriers to Uptake and Potential Intervention Targets[END_REF]. Nevertheless, the effect of CGM in a pediatric population can be the same as in an adult population provided the wearing of the sensor is long enough, as was shown in the SWITCH study (mean wearing of the sensor: 80% of the time, 73% in youths) [START_REF] Battelino | The use and efficacy of continuous glucose monitoring in type 1 diabetes treated with insulin pump therapy: a randomised controlled trial[END_REF].

The role of short-term diagnostic CGM in helping patients to optimize their insulin regimen has not been examined in a large dedicated study. Only two studies reported some treatment changes [START_REF] Boland | Limitations of conventional methods of selfmonitoring of blood glucose: lessons learned from 3 days of continuous glucose sensing in pediatric patients with type 1 diabetes[END_REF] or treatment intensification [START_REF] Kaufman | A pilot study of the continuous glucose monitoring system: clinical decisions and glycemic control after its use in pediatric type 1 diabetic subjects[END_REF] after a short-term CGM course in children. One study reported the effect of advice on bolus timing / use of active insulin after a 3-day masked CGM course in youths [START_REF] Rasbach | Treatment recommendations following 3-day masked continuous glucose monitoring (CGM) in youth with type 1 diabetes[END_REF]. Thus, we designed the DIACCOR Study, which was a national multicenter study aiming to look in real-life conditions at the impact of short-term (7-day) RT-CGM on the insulin treatment strategy and more specifically on the decision for either MDI intensification or switching from MDI to CSII or educational reinforcement. Since the pediatric population differs from adults, with the parents being very much involved in therapeutic decisions and with a strong interaction between the family and the pediatrician, we designed a specific pediatric study and report the results here.

Patients and Methods

2.1.Design of the study

DIACCOR was a French multicenter longitudinal observational study including an adult study [START_REF] Picard | Optimization of Insulin Regimen and Glucose Outcomes with Short-Term Real-Time Continuous Glucose Monitoring in Adult Type 1 Diabetic Patients with Suboptimal Control on Multiple Daily Injections: The Adult DIACCOR Study[END_REF] and the present pediatric study. Investigators were diabetes-specialized pediatricians and were selected out of a national list of physicians with experience in the use of CSII and CGM, whatever their practice (university hospital or non-university hospital).

Pediatricians who accepted to participate enrolled the first three consecutive patients (up to 10 patients) who fulfilled inclusion criteria. Enrolment was competitive up to 150 patients (three to 10 per center) over 8 months and each patient was followed up for 4 months.

2.2.Inclusion and exclusion criteria

Patients ≤ 18 years old could be included if they had uncontrolled type 1 diabetes defined by either an HbA1c value >7.5% and/or a history of SH more than once per year and/or recurrent (>4 per week) documented hypoglycemia (<60 mg/dl) events. SH definition was the one used by the ISPAD. Since any type of hypoglycemia in a child requires assistance, SH in children was defined by the loss of consciousness or the presence of seizures. Diabetes had to be treated with insulin injections (at least twice daily) and patients and their parents had to agree to using a 7-day CGM; they had to be able to fill out a satisfaction questionnaire. Exclusion criteria included patients on CSII, current participation in a clinical trial, and patients who could not complete a 7-day CGM.

2.3.Visits

No specific visit was necessary for the study: Patients were followed up as usual and data were recorded at the study-inclusion visit (M0) and at the follow-up visit 4 months later (M4).

Patients (and their parents) agreed to insert a sensor at both M0 and M4. Sensors used were either Enlite® sensors (with a Medtronic pump as monitor) or Dexcom G4® sensors (with an Animas pump as monitor) according to the physician's decision. A nurse employed by VitalAire France, a homecare provider, instructed the family on sensor use (insertion, calibration, display interpretation including trends) and was in charge of potential technical issues, as we previously reported [START_REF] Picard | Evaluation of the Adherence to Continuous Glucose Monitoring in the Management of Type 1 Diabetes Patients on Sensor-Augmented Pump Therapy: The SENLOCOR Study[END_REF]. There was no alarm setting. The patients and their parents also filled out a satisfaction questionnaire both at M0 and M4 either in the waiting room or at home right after the visit; questionnaires were returned with a prepaid envelope.

An additional visit (M+), 7-15 days after M0 and the first CGM period, could be planned to interpret data and change the treatment for one of three predefined choices according to the pediatrician, the youth, and the parents' shared decision.

2.4.Treatment change

A decision to change treatment was based on several items according to the investigator's evaluation. Eleven main items were given to the investigators to help them identify issues in CGM data. These items were subsequently summarized into five pre-specified categories: hyperglycemia, asymptomatic nocturnal hypoglycemia, glucose variability (including succession of hyperglycemia/hypoglycemia and Somogyi effect), inadequate insulin dosing, and dawn phenomenon. After the baseline CGM, treatment decision was made "in real-life conditions" with the youth and his/her parents, and the patients were included in one of the

2.5.Funding of the study

The promotor of the study was VitalAire France, which did not have any role in data interpretation. Logistical issues were managed by GECEM, a contract research organization.

For each patient, at least one of the parents gave informed consent and was given an information letter. The study did not change the physician-patient relationship as all the included patients fulfilled French recommendations for diagnostic CGM and no specific treatment change occurred. The usual real-life treatment decision was simply given the predefined term of "INT" or "CSII" or "ER" decision. Therefore, there was no need to sign a written consent form. The agreement of the CCTIRS and of the CNIL (agencies for the security and confidentiality of data management) was obtained (Number DR-2014-338).

Statistical analysis

Population sets for this study were total population with all included patients and per protocol population including patients with available baseline CGM data. Statistical analyses were conducted using SAS® software, version 9.1 (SAS Institute, Cary NC, USA). Unless otherwise specified, results are expressed for quantitative data as means ± SD; missing data were excluded. Percentages were determined using the number of responses as the denominators. For the determination of the HbA1c development between inclusion and follow-up visit according to treatment strategy, an analysis of covariance model (ANCOVA) was used allowing for adjustment of initial HbA1c values between the groups. The probability of type 1 error (α) was set at 0.05.

Results

Population of the study

A total of 229 children and adolescents were included in the study by 74 physicians from September 2014 to October 2015. Clinical data were available for 211 patients (92.1%) and the initial CGM was performed on 183 patients (79.9%) (Figure 1). The therapeutic decision was available for 175 patients (76.4%). Demographic characteristics are shown in Table 1.

Age ranged from 3 to 18 years. Comorbidities were present in 18 children, the most frequent being autoimmune thyroiditis (n=8) and asthma (n=3). Among the participating children, seven (3.8%) were in preschool, 48 (26.4%) in elementary school, 76 (41.8%) in junior high school, 42 (23.1%) in high school, and nine (4.9%) in other situations (apprentice or special school).

Among the 110 children (60.1%) who practiced sport outside of school, 70 (38.3%) spent more than 2 h per week in physical activity and 43 (23.5%) were involved in competitions.

The mean HbA1c value at study inclusion was 8.7% ± 1.5%; 82.8% of the children had an HbA1c level >7.5%. Among the children, 12.8% had experienced SH events during the 6 months before study inclusion and 22.2% had recurrent mild hypoglycemia. Diabetic ketoacidosis (DKA) and isolated ketosis had occurred in 8.1% and 11.4%, respectively, of the patients in the previous 6 months.

At M0, diabetes treatment included 3.7 ± 1.0 daily times of injection; 158 children (85.8%) had at least three daily times of injections, 16 (9.1%) counted carbohydrates and 64 (35.0%) occasionally used additional rapid insulin injections. The daily frequency of self-monitoring of blood glucose (SMBG) was 4.3 ± 1.6 with almost 72% of the children testing four times or more per day. Only two patients did not use insulin pens but syringes for injections and all but nine patients (4.9%) used a long-acting analogue as basal insulin.

3.2.CGM data

Sensors were either Enlite® sensors in 62.1% of the children or Dexcom G4® sensors in 37.9%. The mean duration of wearing the diagnostic sensor was 7.7 ± 2.1 days (median 7.0 days, range 1-19). Most of the patients (86.3%) had a dedicated visit (M+) to interpret the CGM data. The most frequent issue revealed by the CGM among the 11 suggested items was postprandial hyperglycemia that was found in 62.8% of the patients (Table 2). When the five prespecified items were considered, hyperglycemia was present in 82.2% of the children, inadequate insulin dosing in 63.9%, asymptomatic nocturnal hypoglycemia in 23.3%, glucose variability in 21.7%, and dawn phenomenon was detected in 18.9% of the patients.

3.3.Treatment strategy after CGM

The therapeutic decision was available for 175 patients, but full data were available only for 168 patients. Changes in insulin doses or regimen were proposed to 158 patients (94.0%). Among them, 139 patients (82.7%) were included in the INT subgroup and 19 patients (11.3%) in the CSII subgroup. Intensification of the insulin regimen in patients on MDI consisted mainly in additional daily injections (≥4 daily in 91.4%) and carbohydrate counting was implemented/reinforced in 11.7% of them. Educational reinforcement without change in insulin regimen occurred in 10 children (6.0%) (ER subgroup).

The therapeutic decisions according to baseline characteristics and CGM data in the three predefined groups are shown in Table 3 andTable 4, respectively. SH or recurrent hypoglycemia history appeared to be more frequent in the CSII subgroup (61.1%) compared with the INT subgroup (30.5%) or ER subgroup (23.6%). The decision for CSII initiation was taken mostly in the presence of chronic hyperglycemia (72%), glucose variability (44.4%), and inadequate insulin dosing (38.9%). The frequency of practicing four or more SMBG tests per day at M0 appeared higher in the 19 children for whom CSII was initiated compared with the 139 children with MDI intensification and the 17 children with no change (83.3%, 71.4%, and 62.5%, respectively). CSII was initiated less often in children who were physically active outside of school (31.6% vs. 68.4%) and a switch to CSII occurred in only one child out of the 70 children physically active more than 2 h per week outside of school.

3.4.Follow-up at 4 months

Among the 183 patients who had the initial CGM, 145 (79.2%) had an M4 visit and 142 (77.6%) a second CGM. M4 was done 161.8 ± 76.2 days after M0 (median: 140 days).

During this period, 14 unscheduled hospitalizations occurred in 13 patients, eight times for diabetes-related events (two ketoacidosis, two hypoglycemia, four uncontrolled diabetes). At M4, a dramatic decrease in asymptomatic nocturnal hypoglycemia was observed (11.1% vs.

23.3% at M0) while the frequency of nocturnal hyperglycemia slightly increased (43.6% vs. 37.8%) (Table 2). Compared with the 6 months before study inclusion, there was a dramatic drop in the percentage of children experiencing SH (3.4% vs. 12.8%) or frequent mild hypoglycemia (6.0% vs. 22.2%) between M0 and M4. The same trend was observed for the percentage of children who experienced DKA and mild ketosis (2.1% vs. 8.1% and 6.9% vs.

11.4%, respectively).

The mean HbA1c level decreased from 8.7 ± 1.5% at M0 to 8.4 ± 1.4% at M4 (p<0.001).

After adjustment to baseline HbA1c values in the INT subgroup (8.7 ± 1.4%), CSII subgroup (8.6 ± 2.0%), and ER subgroup (8.4 ± 1.2%), we found that the HbA1c reduction was significant in the INT subgroup (-0.29%, p=0.009), but not in the CSII or the ER subgroups (-0.48% and -0.42%, p=0.10 and p=0.24, respectively).

Children were satisfied or very satisfied with wearing the sensor, with 93.0%, 100%, and 100% of satisfaction expressed by the children of the INT, CSII, and ER subgroups, respectively. Furthermore, 54.5%, 54.5%, and 50.0% of the children (and 35.1%, 25.0%, and 16.7% of their parents, respectively) from the INT, CSII, and ER subgroups, respectively, wished they could wear a sensor on a long-term basis.

Discussion

While the usefulness of sensor-augmented MDI therapy has been widely demonstrated in T1D, specific issues exist in the pediatric population with potential negative effects of CGM [START_REF] Taddeo | Continuous Glucose Monitoring in Pediatrics: The Gap between Potential Benefits and the Reality of Utility[END_REF]. Interferences of CGM on the quality of life could be a cause for the poor adherence of sensor wear in youth that has been consistently reported. Since adherence is necessary for CGM success, this could be the reason why most of the studies could not show CGM benefits in youths. Nevertheless, the impact on children with uncontrolled T1D on MDI, their parents, and the pediatrician of short-term diagnostic RT-CGM in decision-making had not been considered before this study.

At inclusion in the study, the HbA1c value was consistent with other pediatric studies. In theT1D Exchange Clinic Registry [START_REF] Miller | Current state of type 1 diabetes treatment in the U.S.: updated data from the T1D Exchange clinic registry[END_REF], the mean HbA1c value at enrollment (2010-2012) was 8.3% for 6-12-year-old children and 8.7% for 13-17-year-old teens (n=4,061 and 3,213, respectively). The mean HbA1c value in our population aged 12.2 ± 3.5 years seems comparatively slightly higher (8.7% ± 1.5%), but we selected the children. One specificity of our study is that we selected children with SH and/or uncontrolled diabetes and this translates into SH and DKA reports at M0 (12.8% and 8.1%, respectively) much higher than those in the T1D Exchange Clinic Registry (2-5% in different age groups and 3%, respectively). This selection in diagnostic CGM indication, combined with an "issue guide" provided to the investigator for issue identification, probably played a role in decision-making on the basis of data analysis by the specialist.

The first CGM led to changes in all of the children who were accordingly allocated to one of the three predefined groups. Overall, 82.7% of the youths were allocated to the MDI intensification group (even if most of them injected at least three times daily at M0) that included the implementation/intensification of carbohydrate counting. RT-CGM probably provided a lot more information about glucose patterns to the pediatricians and to the families compared with self-monitoring of blood glucose. Asymptomatic NH was quite frequent at M0 (23.3%), while Ahmet et al. [START_REF] Ahmet | Prevalence of nocturnal hypoglycemia in pediatric type 1 diabetes: a pilot study using continuous glucose monitoring[END_REF] reported an even higher prevalence of 52% asymptomatic NH in children using CGM when using the same glucose threshold. A more than 50% drop in asymptomatic NH occurred between M0 and M4, whereas, simultaneously, there was an increase in nocturnal hyperglycemia of about "only" 15%. This suggests that parents and children learned from the CGM data analysis and probably reduced most often the dose of basal insulin in adequate proportions in children on MDI, while this was considered by the pediatrician in the basal rate setting for children included in the CSII subgroup.

The numbers of children in the CSII and ER subgroups were too small to allow for comparisons per subgroups. This is also probably the reason why the adjusted drops in HbA1c in the CSII and ER subgroups (-0.48% and -0.42%, respectively) were not significant while the decrease in the MDI subgroup (-0.29%) was highly significant (p=0.009).

The trend in the ER subgroup, in the absence of major change in the treatment, suggests that an issue such as abnormal eating behavior [START_REF] Ryan | The prevalence of abnormal eating behaviour in a representative sample of the French diabetic population[END_REF] and/or overcorrection of hypoglycemia and/or inappropriate bolus timing was identified with the short-term CGM sequence and fixed with educational reinforcement.

One of the main reasons for changing treatment was the succession of hyper-and hypoglycemia. Glycemic variability could play an important role in the development of complications in T1D and it appears to be more frequent in youths [START_REF] Forlenza | Ambulatory glucose profile analysis of the juvenile diabetes research foundation continuous glucose monitoring dataset-Applications to the pediatric diabetes population[END_REF]. It was shown that sensor-augmented pump (SAP) therapy improved glucose variability in children compared with MDI [START_REF] Slover | Effectiveness of sensor-augmented pump therapy in children and adolescents with type 1 diabetes in the STAR 3 study[END_REF]. A 1-month CGM use could be helpful in decreasing glucose variability and improving endothelial function [START_REF] Jamiołkowska | Impact of Real-Time Continuous Glucose Monitoring Use on Glucose Variability and Endothelial Function in Adolescents with Type 1 Diabetes: New Technology--New Possibility to Decrease Cardiovascular Risk?[END_REF]. In our study, pediatricians observed a slight decrease in the prevalence of glucose variability on the second CGM compared with the first one. It is impossible to conclude that glucose variability was improved by the 1e-week CGM, but it could have helped. Glucose variability was one of the main reasons that was put forward for switching patients to CSII. CSII was initiated in 19 patients (10.9%) after the first CGM. CSII was shown to improve glucose variability (with a similar HbA1c level) and treatment satisfaction in young patients in transition from pediatric to adult care [START_REF] Maiorino | Treatment satisfaction and glycemic control in young Type 1 diabetic patients in transition from pediatric health care: CSII versus MDI[END_REF]. Furthermore, families who participated in clinical studies evaluating MDI versus CSII usually chose to continue with CSII after the study was completed even when no benefit could be shown with CSII [START_REF] Nahata | Insulin therapy in pediatric patients with type I diabetes: continuous subcutaneous insulin infusion versus multiple daily injections[END_REF]. SAP therapy was also shown to be more effective than sensor-augmented MDI therapy in children in terms of both glucose mean values and glucose variability. Interestingly, only one of the very active youths (>2 h per week of physical activity outside of school) had CSII initiation although the adaptation of the insulin dose with a pump was shown to improve post-exercise glucose control after moderate-to-vigorous exercise in children [START_REF] Moniotte | Outcomes of algorithm-based modifications of insulinotherapy during exercise in MDI vs insulin pump-treated children with type 1 diabetes: Results from the TREAD-DIAB study[END_REF]. Binek et al. [START_REF] Binek | Reasons for the discontinuation of therapy of personal insulin pump in children with type 1 diabetes[END_REF] reported that "difficulties in doing sport" is given as a motive for pump discontinuation in 70% of the children who quit CSII. It is likely that frequent physical activity is one of the reasons for CSII refusal and this should be systematically discussed with the youth and the family.

Regarding sensor wear, it appears that children and teens had very high degrees of satisfaction with CGM. It is probable that most often, parents were worried about this new technology and even if no alarms were set in this study, it was reported that, often, parents have negative emotions against CGM with sleep disturbances, although they recognize the role of CGM in glucose control improvement [START_REF] Barnard | Impact of Type 1 Diabetes Technology on Family Members/Significant Others of People with Diabetes[END_REF]. Things could change with new generations of sensors featuring improved accuracy and no need for confirmatory SMBG [START_REF] Bailey | Clinical Implications of Accuracy Measurements of Continuous Glucose Sensors[END_REF], such as the FreeStyle Libre® that has been positively rated by both children (boys and girls) ≥4 years old and their parents [START_REF] Edge | An alternative sensor-based method for glucose monitoring in children and young people with diabetes[END_REF].

There are limitations to our study. This was a real-life study and thus there was no control group. Furthermore, even if HbA1c variation could be statistically evaluated after adjustment to the baseline value, the difference between the 6-month retrospective frequency of SH or recurrent mild hypoglycemia or ketosis and the 4-month prospective incidence of these issues between M0 and M4 did not allow for any statistical comparison. Finally, the patients included in this study might not reflect all T1D children on MDI as almost three fourths of them had a daily SMBG frequency of ≥4 and they were, moreover, followed up by specialists with experience in both CSII and CGM. Nevertheless, these patients were consecutive patients followed up by pediatricians in different settings and real-life conditions. This study confirms the two mains indications of retrospective CGM as defined in the French position statement [START_REF] Joubert | Société Francophone du Diabète (SFD); Société Française d'Endocrinologie (SFE); EVAluation dans le Diabète des Implants ACtifs Group (EVADIAC). Indication, organization, practical implementation and interpretation guidelines for retrospective CGM recording: A French position statement[END_REF]: HbA1c levels above target and patients unaware of-or suspected of-having frequent/severe hypoglycemia. Miscellaneous indications also include brittle diabetes, help with determining carbohydrate counting parameters, and physical activity. It was also shown that retrospective CGM helps in deciding changes in treatment and results in improvement during subsequent CGM recordings [START_REF] Schaepelynck-Bélicar | Improved metabolic control in diabetic adolescents using the continuous glucose monitoring system (CGMS)[END_REF]. Our study suggests that for the few T1D children and/or parents who refuse to use a sensor on a long-term basis, a short-term use could be very beneficial.

Conclusion

This is the first real-life large study including children with uncontrolled T1D showing that diagnostic RT-CGM helps with treatment strategy by identifying control issues such as glucose variability and by giving tips for diabetes management and insulin treatment intensification. Diagnostic CGM induced changes in treatment and sometimes in CSII initiation. HbA1c improved significantly with MDI intensification and these trends were observed in the few children who switched to CSII or even those without major changes in treatment but with educational reinforcement. Overall, the incidence of SH and DKA dropped dramatically. The indications for diagnostic RT-CGM are not the same as for continuous CGM, but it could help youths who do not want to wear permanent CGM.

Acknowledgments:

This study was sponsored by and funded by VitalAire France. Statistical analyses were performed by GECEM, 85-87 rue Gabriel Peri, 92120 Montrouge, France. Editorial support was provided by Sylvie Picard, MD, PhD (Dijon, France). Funding for this assistance was provided by VitalAire France. The authors are very grateful to the patients and the patients' families for their committed participation in the study; they also thank the nurses from Vitalaire France for their involvement in the study and all the investigators Disclosure statement:

  three predefined subgroups: (a) the INT subgroup was MDI intensification including increases in insulin dosing and/or in the number of daily injections and/or carbohydrate counting initiation or reinforcement; (b) the CSII subgroup included patients who switched to CSII; (c)in the ER subgroup, only educational reinforcement was performed including diet modifications, recommendations for the prevention and correction of hypoglycemia, changes in bolus timing, and intensification of blood glucose monitoring without any change in the insulin regimen. The decision of the inclusion in one of the subgroups was based on the youth's, his/her parents', and the pediatrician's shared decision.

Figure 1 :

 1 Figure 1: Pediatric DIACCOR patient flow chart

Table 1 : Demographic parameters at study inclusion

 1 Unless otherwise specified, values are expressed as mean ± SD

		Total population	Per protocol population
		(n=211)	(n=183)
	Male/female, %	55.2 / 44.8	58.8 / 43.2
	Age (years)	12.2 ± 3.5	12.3 ± 3.4
	Duration of diabetes (years)	4.82 ± 3.58	4.76 ± 3.49
	Weight (kg)	47.6 ± 16.6	48.5 ± 16.2
	Height (cm)	150.6 ± 21.0	151.6 ± 20.8
	HbA1c, %	8.7 ± 1.5	8.7 ± 1.5
	≤ 7.5% / > 7.5% ; %	17.2 / 82.8	16.5/ 83.5
	Daily SMBG tests		4.3 ± 1.6
	< 4 daily / ≥4 daily, %		28.2 / 71.8
	At least 1 comorbidity, %	8.5	8.7
	DKA during the previous 6
	months, Yes, %	8.1	8.7
	Isolated ketosis during the
	previous 6 months, Yes, %	11.4	10.9
	SH during the previous 6
	months, Yes, %	12.8	12.6
	>4 documented mild
	hypoglycemia episodes per	22.2	22.5
	week, Yes, %

SMBG: self-monitoring of blood glucose, DKA: diabetic ketoacidosis, SH: severe hypoglycemia

Table 2 : Results of the inclusion (M0) and 4-month follow-up (M4) CGM data analysis.

 2 Data are expressed as the percentage of patients for whom the parameter was reported by the investigator in the CGM data analysis

	M0	M4

Table 3 : Treatment management according to baseline characteristics

 3

		MDI	CSII	Educational
		intensification	initiation	reinforcement
		(n=139)	(n=19)	(n= 17)
	HbA1c (n=168)
	<7.0% (n=8)	7 (5.3%)	1 (5.6%)	0
	7.0-8.0% (n=48)	33 (24.9%)	8 (44.4%)	7 (41.2%)
	>8.0% (n=112)	93 (69.8%)	9 (50.0%)	10 (58.8%)
	SH history (n=175)
	Yes (n=23)	14 (10.1%)	6 (31.6%)	3 (17.6%)
	No (n=152)	125 (89.9%)	13 (68.4%)	14 (82.4%)
	>4 mild hypo/week (n=163)
	Yes (n=37)	28 (21.5%)	6 (37.5%)	3 (17.6%)
	No (n=126)	102 (78.5%)	10 (62.5%)	14 (82.4%)
	SH OR >4 mild hypo/week
	(n=55)	40 (30.5%)	11 (61.1%)	4 (23.6%)
	No SH AND ≤ 4 mild	91 (69.5%)	7 (38.9%)	13 (76.4%)
	hypo/week (n=111)
	CSII: continuous subcutaneous insulin infusion; SH: severe hypoglycemia, NH: nocturnal
	hypoglycemia; hypo: hypoglycemia

Table 4 : Treatment management according to CGM findings

 4

		MDI	CSII	Educational
		intensification	initiation	reinforcement
		(n=139)	(n=19)	(n= 17)
	Hyperglycemia
	Yes (n=144)	120 (86.9%)	13 (72.2%)	11 (64.7%)
	No (n=29)	18 (13.1%)	5 (27.8%)	6 (35.3%)
	Nocturnal recurrent "hyper"
	Yes (n=66)	54 (39.1%)	7 (38.9%)	5 (29.4%)
	No (n=107)	84 (60.9%)	11 (61.1%)	12 (70.6%)
	Asymptomatic NH
	Yes (n=41)	33 (23.9%)	4 (22.2%)	4 (23.6%)
	No (n=132)	105 (76.1%)	14 (77.8%)	13 (76.4%)
	Glucose variability including
	Somogyi effect
	Yes (n=39)	27 (19.6%)	8 (44.4%)	4 (23.6%)
	No (n=134)	111 (80.4%)	10 (55.6%)	13 (76.4%)
	Inadequate insulin dosing
	Yes (n=112)	96 (69.6%)	7 (38.9%)	9 (52.9%)
	No (n=61)	42 (30.4%)	11 (61.1%)	8 (47.1%)
	Dawn phenomenon
	Yes (n=32)	26 (18.8%)	3 (16.7%)	3 (17.6%)
	No (n=141)	112 (81.2%)	15 (83.3%)	14 (82.4%)

CSII: continuous subcutaneous insulin infusion; SH: severe hypoglycemia; NH: nocturnal hypoglycemia; hyper: hyperglycemia; hypo: hypoglycemia

(Toulouse), Nsota Mbango Jacqueline (Le Mans), Orzechowski Christine (Bry Sur Marne), Paget Corinne (Chambery), Pambou Armelle (Arras), Pantalone Létitia (Pontoise), Pelissier Priscille (St Priest en Jarez), Pla Hélène (La Rochelle), Rougeoreille Charlotte (Melun), Salet Randa (Nîmes), Sarraf Nahla (Cherbourg En Cotentin), Saunier Pascal (Fontainebleau), Staumont Hélène (Quimper), Talvard Maeva (Toulouse), Tiriau Soizic (La Roche Sur Yon), Troller Sophie (La Rochelle), Trollet Tiffany (Sallanches), Ventura Valérie (Palavas Les Flots), Vignon Savoye Capucine (Montfermeil), Wright Catherine (Pringy), Zimmermann Brigitte (Troyes).

Study funded by VitalAire France Abbott, Bayer Diagnostics, Lifescan, Vitalaire, Novo Nordisk, Sanofi, work, EBG reports personal fees from Medtronic, Lilly, VitalAire, IsisDiabete, Dinno Santé outside the submitted work ; EL declares the fees Serono, Vitalaire, Orkin, Lilly, Pfizer, Sandoz, Merck Serono, Ferring and personal fees from Ipsen, Vitalaire, Orkyn, Sanofi, Lilly, outside the submitted work.

SP reports personal fees from Vitalaire, during the conduct of the study, personal fees from Abbott, Bayer Diagnostics, Lifescan, Vitalaire, Novo Nordisk, Sanofi, Animas, and Janssen outside the submitted work, EBG reports personal fees from Medtronic, Lilly, VitalAire, IsisDiabete, Dinno Santé outside the submitted work ; EL declares the fees and payment from Vitalaire for statistical data analysis of the manuscript ; PB reports grants from Serono, Vitalaire, Orkin, Lilly, Pfizer, Sandoz, Merck Serono, Ferring and personal fees from Ipsen, Vitalaire, Orkyn, Sanofi, Lilly, outside the submitted work.