Candice Marion

Philippe Zizzari

Raphael G P Denis

Rim Hassouna

Yacine Chebani

Thierry Leste-Lasserre

Hélène Doat

Gwenaëlle Le Pen

Daniela Cota

Florence Noble

Serge Luquet

Jacques Pantel
email: jacques.pantel@inserm.fr

The GhsrQ343X allele favors the storage of fat by acting on nutrient partitioning

Keywords: Ghrelin, GHSR, Mutant rat, Energy metabolism, Food intake

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

Introduction

The growth hormone secretagogue receptor (GHSR) (Howard, et al. 1996) holds a unique interest as the target of the gut hormone ghrelin [START_REF] Kojima | Ghrelin is a growth-hormonereleasing acylated peptide from stomach[END_REF]), a hormone with key pharmacological properties such as GH release [START_REF] Kojima | Ghrelin is a growth-hormonereleasing acylated peptide from stomach[END_REF], enhanced fat storage and enhanced food intake, mediated by its action on brain energy homeostasis centers [START_REF] Nakazato | A role for ghrelin in the central regulation of feeding[END_REF]Theander-Carrillo, et al. 2006;Tschop, et al. 2000) and hedonic circuits [START_REF] Abizaid | Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite[END_REF][START_REF] Jerlhag | Ghrelin administration into tegmental areas stimulates locomotor activity and increases extracellular concentration of dopamine in the nucleus accumbens[END_REF][START_REF] Naleid | Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens[END_REF].

While the ablation of the Ghsr gene (Ghsr -/-) or of ghrelin-producing cells in mice reported mitigated results on energy homeostasis (Muller, et al. 2015), Ghsr -/-mice failed to enhance locomotor activity similarly to control mice during scheduled feeding [START_REF] Blum | Reduced anticipatory locomotor responses to scheduled meals in ghrelin receptor deficient mice[END_REF][START_REF] Lesauter | Stomach ghrelin-secreting cells as foodentrainable circadian clocks[END_REF]) and to preserve glycemic control during severe caloric restriction (Wang, et al. 2014). The GHSR could therefore play key roles in situations of stress while its role in the fed state remains largely unknown [START_REF] Mani | Ghrelin as a Survival Hormone[END_REF].

The GHSR is a G protein-coupled receptor (GPCR) showing high constitutive activity as documented in cellular (Holst, et al. 2003) and acellular systems (Damian, et al. 2012). Recent observations identified the liver hormone LEAP2 as a novel GHSR ligand (Ge, et al. 2018) with inverse agonist properties (M 'Kadmi, et al. 2019;[START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF]. Therefore, according to its expression pattern in the periphery and in several key brain regions, including the hypothalamus, ventral tegmental area (VTA) or hippocampus (Zigman, et al. 2006), the GHSR could exert its functions according to a complex balance between GHSR ligands, whose accessibility in each GHSR expressing brain structure still needs to be delineated [START_REF] Perello | Brain accessibility delineates the central effects of circulating ghrelin[END_REF]. Overall, these observations, that appear as a game changer [START_REF] Andrews | The next big LEAP2 understanding ghrelin function[END_REF], support an unprecedented refinement for an endocrine system, questioning the therapeutic potential of this GPCR target [START_REF] Al-Massadi | Ghrelin and LEAP-2: Rivals in Energy Metabolism[END_REF]. We reasoned that a preclinical model with shifted canonical GHSR signaling could provide key insights for future drug discovery.

To this purpose, we used a rat model carrying the Ghsr Q343X nonsense mutation that is predicted to cause the deletion of the last 22 amino acids of the GHSR, a domain containing most Ser/Thr phosphorylation sites [START_REF] Bulbul | Food intake and interdigestive gastrointestinal motility in ghrelin receptor mutant rats[END_REF]. Although some studies initially documented altered responses when ghrelin was injected to homozygous mutant rats carrying this allele (Ghsr M/M rats) [START_REF] Bulbul | Food intake and interdigestive gastrointestinal motility in ghrelin receptor mutant rats[END_REF][START_REF] Clifford | Attenuation of cocaine-induced locomotor sensitization in rats sustaining genetic or pharmacologic antagonism of ghrelin receptors[END_REF][START_REF] Mackay | Rats with a truncated ghrelin receptor (GHSR) do not respond to ghrelin, and show reduced intake of palatable, high-calorie food[END_REF], we proposed that the mutant receptor encoded by this mutated allele could enhance GHSR sensitivity to ligands [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]. First, in cellular systems, the GHSR-Q343X receptor shows altered -arrestin recruitment and internalization to the benefit of G protein signaling in response to the agonist. Second, young adult Ghsr M/M rats show increased GH release and chow intake at low doses of the agonist. Third, Ghsr M/M rats develop fat accumulation and insulin resistance with age, an evolutive phenotype consistent with enhanced ghrelin effects [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]). However, the contribution of energy homeostasis to the onset of fat accumulation in Ghsr M/M rats remained unexplored, as well as the physiological consequences of this mutation on food-related behaviors.

In this study, we assessed the consequence of shifting GHSR signaling onto 1) feeding and locomotor response to pharmacological manipulations or feeding challenges, and 2) metabolic efficiency, nutrient partitioning by using an automated food and calorimetric recording system.

Material and methods

Animals

The mutant line of FHH-Ghsr m1Mcwi rats carrying the Ghsr Q343X allele was obtained from the Medical College of Wisconsin (Milwaukee, WI, USA). The homozygous FHH-Ghsr m1Mcwi rats and wild-type littermates are referred to as Ghsr M/M and Ghsr WT/WT rats, respectively. Animals used in this study (187 rats from 7 litters) were obtained from crossing heterozygous rats. Animals were raised four by cages with free access to water and chow diet (A04, SAFE), in a room with controlled temperature (22-24°C) and illumination (12h light/dark schedule with lights on at 7:00 am). The genotype of the rats at the Ghsr locus was determined as previously described [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]. Experiments were performed with male or female rats with ad libitum access to food and water, unless otherwise specified. The procedures involving rats were approved by the ethics committee of animal experimentation of the Université Paris-Descartes.

Analysis of Metabolic efficiency

Calorimetry exploration was performed using 10 week-old male rats at the start of the experiment. Body composition was assessed at the start of the experiment, at the end of baseline, after 24h fasting, and after 48h refeeding (end of the experiment) using an Echo Medical system (EchoMRI 100, Whole Body Composition Analyser, EchoMRI, Houston, USA). Energy expenditure, oxygen consumption and carbon dioxide production, respiratory exchange ratio (RER), food intake and homecage activity were obtained using calorimetric chambers (Labmaster, TSE Systems GmbH, Bad Homburg, Germany) as previously depicted [START_REF] Joly-Amado | Hypothalamic AgRP-neurons control peripheral substrate utilization and nutrient partitioning[END_REF]. Rats were individually housed, fed standard chow and acclimated to the chambers for 48h before experimental measurements. A meal was defined as the consumption of > 0.3 g of food, separated from the next feeding episode by at least 10 mins. To assess metabolic flexibility, all RER data were compiled to obtain relative cumulative frequency curves for Ghsr M/M and Ghsr WT/WT rats. Sigmoidal dose-response curves were fitted to determine EC50 and Hill slopes (indicative of metabolic rigidity) [START_REF] Riachi | Percent relative cumulative frequency analysis in indirect calorimetry: application to studies of transgenic mice[END_REF]).

Hormone measurements

Blood samples were collected from the same cohort of rats in a second experiment performed 3 weeks after the calorimetry exploration. Blood samples were obtained by tail bleeding in ad libitum fed and 24h fasted conditions. Samples for LEAP2 measurements were collected on EDTA and centribuged, and plasma were stored at -80°C until measurements. Sample preparation for ghrelin measurements were performed as previously reported [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]. Rat acyl ghrelin and des-acyl ghrelin were mesured with ELISA kits (Bertin Pharma) and LEAP2 was mesured using an EIA kit (Phoenix Pharmaceuticals).

Statistics

Results are presented as mean ± SEM. Sample size (n) and p values are given in the figure captions.

Statistical analyses were performed using GraphPad Prism® 5.01, SPSS statistics (IBM Inc.) and R software. Differences between 2 groups were determined using non-parametric Mann-Whitney or Wilcoxon tests, or parametric Student's tests, as appropriate. Comparisons of multiple groups were performed using two-way ANOVA, repeated measure ANOVA or ANOVA on aligned rank transformed data (ARTool package) and p value of post-hoc tests were adjusted with the Sidak correction. Covariance analyses (ANCOVA) used the Mouse Metabolic Phenotyping Center web page http://www.mmpc.org/shared/regression.aspx). For all statistical analyses, a p value of less than 0.05 was considered as significant.

Results

Pharmacological probing of central dopaminergic circuits in Ghsr M/M rats reveals enhanced locomotor response to a GHSR agonist but preserved responses to amphetamine or to a DRD2 agonist To examine central dopaminergic circuits in Ghsr M/M rats, locomotion or refeeding response was examined in Ghsr M/M and Ghsr WT/WT rats in response to pharmacologic modulators of dopamine signaling or GHSR agonist. First, we assessed the locomotor response to amphetamine (AMPH), a psychoactive drug known to reverse dopamine transporter activity at post-synaptic target leading to enhances DA release and action (Fig. 1A,B,C). Both experimental groups displayed similar response to the novel environment (Fig. 1A), and to AMPH-induced hyperlocomotion (Fig. 1B,C). Second, GHSR-DRD2 heteromers can produce behavioral response independent of ghrelin binding and correlated to DA function [START_REF] Kern | Apo-ghrelin receptor forms heteromers with DRD2 in hypothalamic neurons and is essential for anorexigenic effects of DRD2 agonism[END_REF]). Ghsr M/M rats model offer a great platform to probe how possible interaction of GHSR and DRD2 receptor might contribute to alter response to DRD2 agonist.To this purpose, the anorexigenic effect of the DRD2 agonist cabergoline was evaluated in Ghsr M/M and Ghsr WT/WT rats.

Cabergoline mediated a potent anorexigenic response in the refeeding of prefasted Ghsr M/M and Ghsr WT/WT animals (Fig. 1D,E). No significant difference was found across genotypes. Third, the pharmacological properties of the GHSR on locomotion were tested in Ghsr M/M and Ghsr WT/WT rats challenged with varying doses of the agonist hexarelin. Repeated measure ANOVA revealed a significant dose x genotype interaction (p<0.01) suggestive of a differential dose-response effect between the two groups of rats (Fig. 1F). Indeed, post hoc analyses showed that the locomotor response to the highest tested dose of hexarelin was twice as high in Ghsr M/M compared to Ghsr WT/WT littermate rats, an observation further confirmed in the analyses of the response to the highest hexarelin dose (Fig. 1G,H).

Overall, these observations, while supporting enhanced GHSR responsiveness in Ghsr M/M rats, also rule out gross abnormalities in the dopaminergic system of these rats.

Ghsr M/M rats show unaltered spontaneous conditioning and motivation for sucrose but accelerated performance in operant system

The former results indicated that relative to their body weight, chow consumption was comparable between Ghsr M/M and Ghsr WT/WT rat, while showing enhanced sensitivity to endogenous ghrelin [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]. We hypothesized that these animals might show improved consumption and/or motivation for palatable food. To test this, we took advantage of the spontaneously high preference for sucrose in the fawn hood strain (Tordoff, et al. 2008) to investigate the spontaneous consumption and reinforcing properties of sucrose in Ghsr M/M and Ghsr WT/WT rats using free choice and instrumental conditioning paradigms. In a two-bottle choice between a sucrose solution and water, both Ghsr M/M and Ghsr WT/WT male littermates displayed similar levels of consumption throughout days, with a strong, sustained preference for the sucrose solution over water (Fig. 2A). For instrumental conditioning paradigms, we focused our interest on female rats who successfully achieved conditioning without requiring calorie restriction, a manipulation known to interfere with ghrelin endogenous tone. During conditioning sessions, ad libitum fed Ghsr M/M and Ghsr WT/WT rats achieved success criterion during the last 2 sessions of each Fixed Ratio (FR) schedule (Fig. 2B upper panel; repeated measure ANOVA; session effect; p<0.001).

Total responding levels were similar between Ghsr M/M and Ghsr WT/WT rats on the Progressive Ratio (PR) PR schedule, and for both groups, nose-poking activity on the inactive, dummy target was close to zero, indicating specific, goal-oriented responding at the active nose-poking hole. Interestingly, at 5 mins after the beginning of sessions, Ghsr M/M rats had significantly achieved more responses than Ghsr WT/WT rats, starting from the 3 rd FR3 session (Fig. 2B lower panel), although the total number of responses was comparable between Ghsr M/M and Ghsr WT/WT rats throughout the FR and PR schedules. This significant difference in performing speed was also seen 15 mins after the beginning of the session (Fig. 2B lower panel). Overall, ad libitum fed Ghsr M/M rats showed preference or consumption for palatable food similar to Ghsr WT/WT rats, but performed faster to obtain rewarding food in an operant nose-poking task.

Ghsr M/M display preserved food anticipatory activity in response to scheduled feeding.

Previous investigations revealed that Ghsr M/M rats preserved better their body weight and glycemia in scheduled restricted feeding conditions [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF], however whether this condition also affected other behavioral response related to food-seeking response remained unexplored. To test this, two paradigms differing on the kind of locomotor activity measured (wheel running or ambulatory locomotion) were used. In the first experiment, Ghsr M/M and Ghsr WT/WT littermate rats habituated to a single housing cage with free access to running wheels were put on a restricted feeding schedule.

Unexpectedly, during the ad libitum feeding period, Ghsr M/M rats showed decreased running activity during the first hour of dark phase (Fig. 3A). At the beginning of caloric restriction, wheel running activity was increased in both groups of rats and the activity of Ghsr M/M rats no longer differed from that of Ghsr WT/WT rats (Fig. 3B). Running activity was further enhanced at the end of the caloric restriction and the activity pattern was re-distributed with maximal activity levels in anticipation to dark phase and meal access (Fig. 3C). Ghsr M/M and Ghsr WT/WT rats displayed comparable levels of daily activity, as well as similar food anticipatory behavior (Fig. 3C). Besides, throughout caloric restriction Ghsr M/M rats consumed similar amounts of food compared to Ghsr WT/WT littermates (Fig. 3D) but were better able to maintain their body weight (Fig. 3E) and glycemia (Fig. 3F). To verify that food anticipatory activity was indeed unaltered in Ghsr M/M rats while avoiding the potentially confounding effect of wheel running, which is rewarding by itself, we used an alternative paradigm in which another cohort of rats at same age were put on a 4h restricted feeding schedule and recorded daily in actimetry cages during the 2 hours preceding food access. In this setting, Ghsr M/M and Ghsr WT/WT rats developed food anticipatory activity to comparable levels (Fig. 3G), while chow intake and body weight were similar across genotypes (Fig. 3H,I). Altogether, Ghsr M/M rats therefore show anticipatory activity and food consumption similar to Ghsr WT/WT littermates on a restricted feeding schedule, suggesting that the Ghsr Q343X mutation does not affect behavioral anticipation of food.

Ghsr M/M rats display increased body mass and similar body composition at 12 weeks of age

In order to determine whether alteration in energy homeostasis precede the development of insulin resistance in 6 month-old rats [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF], metabolic efficiency was assessed using indirect calorimetry in asymptomatic mutant rats in response to nutritional manipulation. Ghsr M/M and Ghsr WT/WT rats entered in metabolic cages at similar age (12.3±0.2 and 12.0±0.3 week-old, respectively). Upon entry, Ghsr M/M rats were on average 7% heavier than Ghsr WT/WT littermates, but proportions of fat and lean masses relatively to total body mass were comparable in Ghsr M/M and Ghsr WT/WT rats (Fig. 4A). Across the experiment, Ghsr M/M and Ghsr WT/WT animals similarly lost and regained total body mass, fat mass and lean mass upon fasting and refeeding, respectively (Fig. 4B). Thus, in young adult rats, the Ghsr Q343X mutation resulted in higher body weight, with increases in both fat and lean masses in proportion to total body mass, suggesting an aggravation of the phenotype later with age, which is in line with similar blood glucose levels between genotypes in all feeding conditions (Supplementary Fig. 1).

As shown Fig. 4C, Ghsr M/M and Ghsr WT/WT animals showed comparable 24h patterns of activity across baseline, fasting and refeeding, both in light and dark phases. In both groups of rats, fasting increased homecage activity, while refeeding returned it to basal level (Fig. 4D). As expected, the feeding pattern post-fast significantly differed from the ad libitum pre-fast condition for both groups of rats (Fig. 4E), with no differences across genotypes regarding 24h intake (Fig. 4F). Furthermore, the diurnal and nocturnal meal parameters across feeding conditions were comparable between Ghsr M/M and Ghsr WT/WT rats, as measured by mean meal size, total time spent eating, number of meals, meal duration, intermeal intervals and ingestion rate (Supplementary Fig. 2). Not surprisingly, both groups of rats showed similar diurnal and nocturnal levels of energy expenditure characterized by decreased energy expenditure during fasting, while there was a general tendency to return to its basal level upon refeeding (Fig. 4G). Analyses of 24h energy expenditure revealed no differences between genotypes in the ad libitum feeding condition, but a trend to decreased energy expenditure was observed in Ghsr M/M rats during fast and refeeding conditions (Fig. 4H). Estimated resting metabolic rate was also comparable for Ghsr M/M rats and their Ghsr WT/WT littermates (9.1±0.4 kcal/h/lean mass and 9.4±0.3 kcal/h/lean mass, respectively). Altogether, these results suggest that in young adult rats, the Ghsr Q343X mutation does not alter daily locomotor activity, caloric intake, meal patterns or energy expenditure in conditions of ad libitum access to food as well as short-term food deprivation.

Ghsr M/M rats exhibit metabolic fuel preference towards carbohydrates

As Ghsr M/M rats showed increased body weight without any major changes in energy intake or expenditure compared to their Ghsr WT/WT littermates, we then investigated if Ghsr M/M and Ghsr WT/WT rats displayed differences in utilization of metabolic substrates, by exploring RER in baseline, fasted and refed conditions (Fig. 5A,B,C). In all nutritional states, mean 24h RER was higher in Ghsr M/M rats compared to Ghsr WT/WT littermates (Fig. 5D), indicating a slight but sustained increase in carbohydrate utilization as energy substrate, at the expense of fat, in Ghsr M/M rats compared to Ghsr WT/WT rats. However, qualitative RER variations across the experiment were similar in Ghsr M/M and Ghsr WT/WT animals, with RER decreasing during fasting compared to ad libitum basal feeding (Fig. 5A,B,D), and returning to baseline levels during the first 24h of refeeding (Fig. 5C,D). Separate analyses of light and dark phases indicated that, during light phase, Ghsr M/M rats had in all conditions a higher RER than Ghsr WT/WT rats (~2% in the fed state) (Fig. 5E) which is not the case during the dark phase (Fig. 5F). Thus, Ghsr M/M compared to Ghsr WT/WT rats presented an overall decreased use of lipids and increased use of carbohydrates as energy substrates illustrated further by decreased cumulative fat oxidation across feeding conditions (Supplementary Fig. 3A,B,C,D). In order to explore metabolic flexibility, RER data were compiled to obtain relative cumulative frequency curves for Ghsr M/M and Ghsr WT/WT rats. Comparison of the fits revealed a right shift of the curve for Ghsr M/M compared to Ghsr WT/WT rats (Supplementary Fig. 3E), indicating that RER distribution of the Ghsr M/M group is narrower and skewed towards higher values compared to the Ghsr WT/WT group. Moreover, 1/Hill slope was lower for Ghsr M/M rats compared with Ghsr WT/WT rats indicative of decreased metabolic flexibility in Ghsr M/M rats.

Ghsr M/M rats show enhanced LEAP2:acyl ghrelin molar ratio but unaltered energy or glucose homeostasis markers centrally or in the periphery Then, blood concentrations of GHSR hormones (i.e. LEAP2 and acyl-ghrelin) were assessed in satiated and fasted states using a subgroup of these same experimental rats (Fig. 5). The two-way ANOVA performed on circulating LEAP2 levels showed a significant genotype x feeding condition interaction (p < 0.01) revealing significantly higher LEAP2 concentrations in Ghsr M/M compared to Ghsr WT/WT littermates rats in the fed state only (Fig. 5G). The same analysis performed on circulating acyl ghrelin levels also showed a significant genotype x feeding condition interaction (p < 0.05) revealing this time lower circulating acyl ghrelin levels in Ghsr M/M as compared to Ghsr WT/WT rats only in the fasting state (Fig. 5H).

Additionally, des-acyl ghrelin levels showed a pattern of response similar to acyl ghrelin (data not shown). When these same data were analyzed using a three-way ANOVA, a significant genotype x feeding condition x hormone interaction (p<0.05) was observed, illustrating a biologically significant interplay between GHSR hormones across feeding conditions in Ghsr M/M rats. Furthermore, Fig. 5I shows that this interplay benefits LEAP2 over acyl-ghrelin levels in Ghsr M/M rats compared to Ghsr WT/WT rats as suggested by increased LEAP2:acyl-ghrelin molar ratio in the former. Finally, to rule out energy or glucose homeostasis abnormalities at gene expression level in asymptomatic Ghsr M/M rats, the expression of selected genes was compared in Ghsr M/M and Ghsr WT/WT rat tissues. These investigations essentially revealed no significant differences across the panel of selected genes between groups (Fig. 6).

Nevertheless, the Ghsr gene showed enhanced expression in the hypothalamus of Ghsr M/M compared to Ghsr WT/WT rats, an observation that specifically matched with the Ghsr1a isoform (Fig. 6A). Despite the low expression level of Ghsr in the peripheral tissues tested, the enhanced sensitivity of the digital PCR technique disclosed similar Ghsr gene expression across groups in the soleus muscle (Fig. 6H). Overall, when still asymptomatic regarding body composition or disordered gene expression markers, Ghsr M/M rats may nevertheless show altered GHSR markers (hormones and gene expression).

Discussion

Recent observations suggest that the GHSR is a constitutively active GPCR endowed of a sophisticated tuning involving by a balance of endogenous ligands (Ge et al. 2018;[START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF]. Therefore, shifting GHSR canonical signaling could provide unique insights for future drug discovery. This is the case of the functionally significant Ghsr Q343X mutation, whose homozygous rat carriers display enhanced responsiveness to GHSR agonists, enhanced fat accumulation, and insulin resistance [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]. The present study now shows that Ghsr M/M rats specifically display an enhanced locomotor response to a GHSR agonist while responses using dopaminergic drugs are seemingly unaltered.

Similarly, spontaneous consumption and conditioning for sucrose appeared not to be impacted by the Ghsr Q343X mutation, nor did food anticipatory activity. Ghsr M/M rats, prior to fat accumulation and insulin resistance, show a shifted fuel preference towards carbohydrates at early adulthood. In contrast, Ghsr M/M rats did not show obvious qualitative or quantitative alterations of energy intake, energy expenditure or locomotion. Altogether, the present study supports the feasibility of biasing GHSR signaling to the benefit of the storage of fat by acting preferentially on nutrient partitioning.

The present study provides novel insights into the relationship between GHSR signaling and metabolic fuel preference. Indeed, Ghsr M/M rats showed a higher RER than Ghsr WT/WT littermates, indicating a shift in metabolic preference towards decreased use of fat. It is noteworthy that increased RER in Ghsr M/M rats was essentially observed during light phase in the ad libitum condition (physiological fasting) and during early fasting and early refeeding. These observations are therefore in line with acute and chronic pharmacological studies showing that supraphysiological levels of acyl ghrelin elevates RER, indicating shifted metabolic fuel preference towards enhanced carbohydrate utilization, while energy expenditure and home cage activity are unaffected [START_REF] Currie | Integrative and Comparative Physiology 289 R353-R358. receptor (GHSR) gene mutations in Japanese subjects with short stature[END_REF]Theander-Carrillo et al. 2006;Tschop et al. 2000). Overall, based on the assets of the Ghsr Q343X model enhancing ghrelin responsiveness [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF], the present data support a physiological role for GHSR signaling in regular diet conditions to promote fat storage and preservation by acting on nutrient partitioning, a mechanism that might be, at least in part, centrally mediated (Joly-Amado et al. 2012).

Metabolic characterization of Ghsr M/M and Ghsr WT/WT littermates explored herein supports a specific alteration of substrate balance (decreased fat oxidation) associated the Ghsr Q343X mutation, rather than a modification of total energy intake or expenditure. Indeed, as measured in freely behaving conditions, Ghsr M/M and Ghsr WT/WT littermate rats showed similar chow intake, feeding patterns and energy expenditure. Although the latter did tend to decrease in Ghsr M/M rats compared to Ghsr WT/WT rats in situations of energy deficit, it did not differ in the fed condition, thus excluding a key role of GHSR signaling in energy expenditure control. Interestingly, young adult Ghsr M/M rats showed increased body weight compared to their Ghsr WT/WT littermates, but displayed equivalent proportions of fat and lean masses relatively to total body mass, comparable fasting glycemia as well as similar expression of several molecular markers of energy and glucose homeostasis in the hypothalamus and several peripheral tissues. In comparison, as exemplified in our prior study [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF], older Ghsr M/M rats showed enhanced body weight and adiposity and decreased glucose tolerance, suggesting that the phenotype of these rats worsen with age, which is consistent with studies in Ghsr -/-mice involving the GHSR in metabolic aging (Lin, et al. 2011;Ma, et al. 2011). Indeed, as they are aging, Ghsr -/-mice fed on a standard diet display reduced obesity and improved insulin sensitivity compared to Ghsr +/+ littermates, a phenotype mirroring that of adult Ghsr M/M rats. In contrast to the mechanism described in Ghsr M/M rats (lower fat oxidation associated with development of adiposity), while RER of 1 year-old mice was similar amongst genotypes, it was increased in 2 year-old Ghsr -/-mice compared to Ghsr +/+ littermates. It was suggested that increased RER in Ghsr -/-mice could be related to their lean phenotype and improved insulin sensitivity [START_REF] Lin | Ablation of ghrelin receptor reduces adiposity and improves insulin sensitivity during aging by regulating fat metabolism in white and brown adipose tissues[END_REF]. Altogether, the phenotype of Ghsr M/M rats and Ghsr -/-mice seems to implicate GHSR signaling in age-associated adiposity and insulin resistance. In addition, we speculate that enhanced acyl ghrelin sensitivity in Ghsr Q343X rats illustrates how low fat oxidation may contribute to the occurrence of overweight (Galgani and Ravussin 2008).

The analysis of circulating levels of GHSR hormones performed herein provide a new perspective regarding the Ghsr Q343X model. The present study confirmed our previous observation that acyl ghrelin levels are similar among groups, but increased less during fasted or negative energy balance states in Ghsr M/M rats compared to their Ghsr WT/WT littermates [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]. Second, the LEAP2 assay disclosed increased levels in Ghsr M/M rats over Ghsr WT/WT rats in the fed condition while these were similar in fasted rats. Altogether, the LEAP2:acyl-ghrelin molar ratio was significantly enhanced across feeding conditions in Ghsr M/M rats compared to Ghsr WT/WT rats, consistent with a concerted regulation of these two hormones across feeding conditions. This could be interpreted as an adaptive response to the enhanced GHSR sensitivity in Ghsr M/M rats. Interestingly, enhanced LEAP2:acyl-ghrelin molar ratio was recently documented in obesity conditions in mice and human patients [START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF]). In our case, however, the 12-weeks old Ghsr M/M rats did not show changes in fat mass or glycemia. Therefore further studies are needed regarding the LEAP2-acyl ghrelin balance in rat carriers of the Ghsr Q343X allele. Finally, these observations may illustrate an evolutive biological state of so called ghrelin resistance in Ghsr M/M rats, a condition documented in obesity [START_REF] Briggs | Diet-induced obesity causes ghrelin resistance in arcuate NPY/AgRP neurons[END_REF]Zigman, et al. 2016). We speculate that this aggravating state could participate in the sometime contradictory pharmacological GHSR responses documented in adult Ghsr M/M rats [START_REF] Bulbul | Food intake and interdigestive gastrointestinal motility in ghrelin receptor mutant rats[END_REF][START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF][START_REF] Clifford | Attenuation of cocaine-induced locomotor sensitization in rats sustaining genetic or pharmacologic antagonism of ghrelin receptors[END_REF][START_REF] Mackay | Rats with a truncated ghrelin receptor (GHSR) do not respond to ghrelin, and show reduced intake of palatable, high-calorie food[END_REF].

The results obtained herein using pharmacological tools refine and further support previous observations on the mechanism-of-action of the Ghsr Q343X mutation in rat. First, enhanced locomotor response to the GHSR agonist hexarelin in Ghsr M/M rats may suggest increased dopaminergic responsiveness compared to Ghsr WT/WT littermates. Former results using dose-responses of ghrelin or hexarelin already showed improved GH release and 4h chow intake, observations indicative of enhanced GHSR responsivity in the hypothalamus [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]. Nevertheless, in contrast to these former results, obtaining a significant locomotor response required the highest dose of the injected agonist suggesting that this response may be non-physiological. In sum, these experiments are consistent with the hypothesis that the Ghsr Q343X mutation, that results in G protein biased signaling in response to agonist in cellular systems [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF], could recapitulate a gain-of-function mutation in the GHSR. Additionally and as disclosed herein, the enhanced expression of Ghsr1a in the hypothalamus but not in a peripheral tissue of young adult Ghsr M/M rats, could also be considered as a possible contributing factor, although this requires further investigations. Second, pharmacological challenges designed to probe dopaminergic circuits using a DAT blocker (amphetamine) or a DRD2 agonist (cabergoline) revealed no difference across Ghsr M/M and Ghsr WT/WT rats. These observations therefore suggest that the Ghsr Q343X allele has no obvious direct or indirect effect regarding both of these pharmacological responses, involving enhanced extracellular dopamine tone (amphetamine) or DRD2-GHSR heterodimers (cabergoline) [START_REF] Kern | Apo-ghrelin receptor forms heteromers with DRD2 in hypothalamic neurons and is essential for anorexigenic effects of DRD2 agonism[END_REF]. Interestingly, rare human missense or nonsense GHSR variants segregating with short stature or GH deficiency, presumed to be loss-of-function mutations on the basis of their in vitro mechanism of action, were documented (Inoue, et al. 2011;[START_REF] Pantel | Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature[END_REF][START_REF] Pantel | Recessive isolated growth hormone deficiency and mutations in the ghrelin receptor[END_REF][START_REF] Pugliese-Pires | Novel Inactivating Mutations in the Growth Hormone Secretatogue Receptor Gene (GHSR) in Patients with Constitutional Delay of Growth and Puberty[END_REF], including the GHSR A204E mutation that specifically alters constitutive activity in vitro. Just recently, a knock-in mice model expressing this mutation showed altered GH release, food intake and glycemic control (Torz, et al. 2020), therefore demonstrating that the GHSR A204E disease-causing mutation is related to a partial impairment of GHSR functioning. This kind of genetic defect mirrors the mechanism of the present Ghsr Q343X mutation in rat, documenting enhanced GHSR function both in vitro and in vivo. Altogether, Ghsr mutant models appear as very relevant tools to probe the significance of GHSR signaling in vivo, more especially since this constitutively active GPCR was recently established as the key target of both the agonist hormone ghrelin and LEAP2, an endogenous ligand with inverse agonist properties (M' Kadmi et al. 2019;[START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF].

Using the Ghsr Q343X model to probe food anticipatory activity, Ghsr M/M and Ghsr WT/WT rats were found to have similar running activity in anticipation of food, suggesting that the Ghsr Q343X mutation does not impact the development nor expression of food anticipatory activity (FAA). Acyl ghrelin has been proposed to be a food-entrainable oscillator that participates in food anticipatory activity in rodents put on a restricted feeding schedule [START_REF] Laermans | Role of the clock gene Bmal1 and the gastric ghrelinsecreting cell in the circadian regulation of the ghrelin-GOAT system[END_REF][START_REF] Lesauter | Stomach ghrelin-secreting cells as foodentrainable circadian clocks[END_REF]). Furthermore, when mice are put on a restricted feeding schedule, Ghsr knock-out results in attenuated FAA [START_REF] Blum | Reduced anticipatory locomotor responses to scheduled meals in ghrelin receptor deficient mice[END_REF]Davis, et al. 2011;[START_REF] Lesauter | Stomach ghrelin-secreting cells as foodentrainable circadian clocks[END_REF]) and reduced activation of several hypothalamic and midbrain nuclei prior to food access [START_REF] Lamont | Ghrelin-deficient mice have fewer orexin cells and reduced cFOS expression in the mesolimbic dopamine pathway under a restricted feeding paradigm[END_REF], altogether supporting that GHSR signaling plays a role in food anticipation. FAA was suggested to have a "go, no-go" property [START_REF] Lesauter | Stomach ghrelin-secreting cells as foodentrainable circadian clocks[END_REF]), therefore, while complete removal of the GHSR results in delayed onset of FAA, the presence of functional canonical acyl ghrelin-GHSR signaling in Ghsr M/M rats, albeit enhanced [START_REF] Chebani | Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite[END_REF]), might produce "go" decisions with probabilities similar to the wild type GHSR without affecting FAA onset. Of note, Ghsr M/M rats were previously reported to have decreased FAA as assessed by total number of infrared bream breaks during the 2 hours preceding food access [START_REF] Mackay | Rats with a truncated ghrelin receptor (GHSR) do not respond to ghrelin, and show reduced intake of palatable, high-calorie food[END_REF], a result that is not supported by present observations using two different paradigms (running wheels and actimetry cages).

However, in this former study, FAA was not normalized to total 24h activity levels, and the observed decrease in FAA is likely to be the result of the general reduction in activity levels related to the metabolic phenotype of Ghsr M/M rats, rather than a specific attenuation of food-oriented anticipatory activity.

Taking advantage of the high preference for sucrose of the Fawn hood strain (Tordoff et al. 2008) to probe spontaneous phenotypes in ad libitum fed Ghsr M/M and Ghsr WT/WT rats, the present study disclosed 1) similar consumption of sucrose in a two-bottle choice paradigm, suggesting that the hedonic perception of sucrose is not altered, and 2) similar levels of total nose-poke responses for sucrose in an instrumental task, supporting similar motivation to obtain food rewards. Thus far, pharmacological studies support that acyl ghrelin-GHSR signaling modulates the appetitive properties of palatable food. Indeed, peripherally administered acyl ghrelin promotes, whereas GHSR antagonist JMV2959 reduces, consumption and motivation to obtain palatable food in ad libitum-chow fed rodents [START_REF] Landgren | The ghrelin signalling system is involved in the consumption of sweets[END_REF][START_REF] Perello | Ghrelin increases the rewarding value of high-fat diet in an orexindependent manner[END_REF]Skibicka, et al. 2012), effects that are essentially reproduced by i.c.v., intra-VTA or intra-ventral hippocampus (vHPC) administration of acyl ghrelin or JMV2959 [START_REF] Kanoski | Ghrelin signaling in the ventral hippocampus stimulates learned and motivational aspects of feeding via PI3K-Akt signaling[END_REF][START_REF] Skibicka | Ghrelin directly targets the ventral tegmental area to increase food motivation[END_REF]Skibicka et al. 2012). However, the physiological significance of these effects is still unclear, as Ghsr -/-, Ghrl -/-and Goat -/-mouse models rarely showed results supporting altered hedonic feeding behaviors when animals are explored in the fed state (Davis, et al. 2012;Disse, et al. 2010;[START_REF] Lockie | Diet-induced obesity causes ghrelin resistance in reward processing tasks[END_REF]. In the physiological setting of the present study, it is interesting to note that Ghsr M/M rats, considered with enhanced response to endogenous ghrelin, did not produce an increase in the number of responses over Ghsr WT/WT rats, but had a subtler effect on the performing speed of the animals, which was increased. This observation may suggest enhanced impulsivity to obtain food rewards. Interestingly, ghrelin injected i.c.v. or into the VTA of rats was indeed recently shown to increase impulsive behavior to obtain palatable food [START_REF] Anderberg | The Stomach-Derived Hormone Ghrelin Increases Impulsive Behavior[END_REF]. Overall, the observation supporting a possible effect of the Ghsr Q343X mutation with qualitative rather than a quantitative modulation of spontaneous operant responding for sucrose, at least in female rats, is of particular interest and needs to be clarified further, keeping in mind possible confounding factor such as the current rat strain as well as the metabolic phenotype of aged Ghsr M/M rats.

The present study shows limits regarding putative sex differences associated with the Ghsr Q343X mutation. First, the present calorimetry study only focused on male rats. While female Ghsr M/M rats developed overweight with age with a timing similar to male rats (our former study), a study specifically investigating the energy intake, energy expenditure or RER parameters in female animals is mandatory in order to delineate possible gender differences. Indeed, female animals in mice or rat are known to gain less weight than males in HFD models, and these observations are known to be contributed, at least in part, to differences in sensitivity to energy homeostasis hormones such as leptin or ghrelin. More specifically, ghrelin sensitivity was shown to be modulated by oestrogens [START_REF] Clegg | Estradiol-dependent decrease in the orexigenic potency of ghrelin in female rats[END_REF]. Second, in the reward experiments using sucrose, while female and male Ghsr M/M rats show similar responses in the 2-bottle choice paradigm, only female rats succeeded the operant conditioning steps, precluding the analysis of any possible specific effect of the mutation in male Ghsr M/M rats.

Altogether, the present study strengthens the observations that Ghsr M/M rats may specifically show enhanced responsivity to GHSR agonist in vivo. In the fed or fasted conditions, young adult Ghsr M/M rats did not show obvious feeding or locomotor alterations but showed shifted fuel preference towards carbohydrates, therefore providing a possible mechanism to the enhanced fat accumulation documented in these rats later with age. Finally, these data also support the feasibility of tricking GHSR signaling to the benefit of fat storage by acting preferentially on nutrient partitioning. Supplementary Figure 3: Decreased fat oxydation in Ghsr M/M rats across feeding conditions. (A-C) Daily pattern of fat oxydation during adlibitum feeding (A), 24h fasting (B) and 24h refeeding (C). Data represent mean ± SEM. (D) 24-h cumulative fat oxydation (n=12/genotype). Data were analyzed by 2-way ANOVA (E) Relative cumulative frequency curves for ad libitum fed Ghsr M/M and Ghsr WT/WT rats (dots) and curve fits (full lines) reveal higher EC 50 (0.947 vs. 0.933) and a lower 1/Hill slope value (0.067 vs. 0.074) for Ghsr M/M rats compared to Ghsr WT/WT rats (F-test; p<0.001 for both parameters). * p<0.05; *** p<0.001.

Material and methods -Supplementary file

Amphetamine-induced locomotion

A group of experimentally naive 10 week-old male rats was used to assess the locomotor response to a novel environment and to the injection of the dopamine enhancing drug amphetamine. Rats were habituated to a locomotor cage (Imetronic, Bordeaux, FR) for 60 mins on 2 consecutive days. The next 2 days, groups of Ghsr M/M and Ghsr WT/WT rats were injected i.p. with amphetamine (2 mg/kg) while a control group of Ghsr heterozygous rats was injected i.p. with saline.

Cabergoline-induced anorexigenic response

Prior to the food intake experiment, 7 month-old female Ghsr M/M and Ghsr WT/WT rats were habituated to single housing for at least 1 week. Free access to chow diet was removed at 6:00 pm for a 16h fast. Rats were then s.c. injected with the dopamine receptor D2 (DRD2) agonist cabergoline (0.5 mg/kg, Tocris) or saline at 11:00 am. Each animal received both treatments in a crossover fashion, with a 2week washout period between treatments.

Hexarelin-induced locomotion

On the first day of each experiment, experimentally naive 5 month-old male rats were individually put in an open field arena (Med Associates Inc., Vermont, USA) with clean bedding. Room illumination was set at 15 lux. Recording sessions began each day at 8:30 am. Rats were habituated to the apparatus on 2 consecutive days for 45 min sessions. The next day, rats were injected by injected s.c. with saline solution (100 µL/100mg). Every other day, rats were challenged with varying doses of hexarelin (10 -300 nmol/kg, Sigma) in the same setup, using a random crossover design.

Sucrose two-bottle choice

Ad libitum fed 7 month-old male rats were habituated to single housing and drinking from 2 bottles of water for at least 1 week prior to the beginning of the experiment. On 7 consecutive days they had access to a 0.75% sucrose solution and water for 1h, starting at 10:00 am. The position of the sucrose bottle was switched every day to prevent habituation. Bottles were weighed before presentation and again at the end of the 1h test to assess fluid intakes.

Operant responding for sucrose pellets Operant chambers (Med Associates Inc., Vermont, USA) comprised 2 open nose-pocking niches, one designed as the active target (triggering reward delivery) and the other as the inactive, dummy target. Entry into the active nose-poking hole was rewarded by the delivery of a 45 mg sucrose pellet (Dustless Precision Pellets®). Entries into the inactive nose-poking hole had no consequence. Houselight was on throughout the session. A light was switched on in the active nose-poking hole or food magazine to cue target or reward availability, respectively.

Ad libitum fed 7 month-old female rats were trained on fixed ratio (FR) schedules, starting with FR1 (1 nose-poke required to obtain 1 pellet), then on FR3 (3 nose-pokes to obtain 1 pellet) and finally FR5. A training session ended after 30 mins or when the rat had obtained 50 pellets (success criterion). Rats were kept on each FR schedule for 4 consecutive sessions, at the end of which all animals had reached the success criterion. After training, the rats underwent consecutive test sessions on a progressive ratio (PR) schedule to assess their motivation to work for the food reward according to the usual exponential formula (N = 5 * exp(0.2N) -5). The session ended when rats had failed to obtain a pellet for 30 consecutive minutes.

Running wheels 7 month-old male Ghsr WT/WT and Ghsr M/M rats matched for body weight were individualized at least 1 week prior to the experiment. Body weight, chow intake and wheel running behavior were monitored daily for 13 days under ad libitum feeding conditions until daily running levels had stabilized. On the last baseline day, chow intake was removed at 1:00 pm, and rats were fasted for 24h. On the following days, rats had free access to chow 2h/day, from 1:00 to 3:00 pm. During food access, running wheels were blocked to make sure the rats would feed and not run. The rats had free access to the running wheel for the 22 remaining hours. Every quarter of wheel turn was detected and recorded using a Matlab program. Raw data were converted to numbers of wheel turns in 1h bins using a dedicated R program. Chronic food restriction lasted for 15 days before rats were euthanized.

Food anticipatory activity assessed in open field arena 7 month-old male rats were individualized at least 1 week prior to the experiment. On the first session, locomotor activity was recorded in the open field arena for 2 hours before rats were returned to their homecage where food had been removed. During the 20 following days, rats were recorded daily in the same arena for 2h (bedding left in place) before being returned to their homecage and having access to chow diet for 4h. Body weight and chow intake were assessed daily throughout the experiment.

Quantitative real-time PCR analysis

Analyses were performed on snap frozen tissues stored at -80°C and collected during the light phase in libitum fed rats. Tissues samples (hypothalamus, adipose, muscle and liver) were homogenized in Tri-reagent (Euromedex, France) and RNA was isolated using a standard chloroform/isopropanol protocol. Reverse transcriptase reactions were performed from 2 µg of total RNA using Maxima Reverse Transcriptase (Fisher Scientific), primed with oligo-dT primers and Random Hexamer Primer (Fisher Scientific). Real-time PCR was performed on a LightCycler 480 Real-Time PCR System (Roche Diagnostics) using transcript-specific primers (600 nM), LightCycler 480 SYBR Green I Master (Roche Diagnostics) and cDNA (4 ng) in a final volume of 10 µl. Relative expression of each gene was normalized respect to 2 different endogenous housekeeping control genes depending on the tissue and determined using RefFinder analysis. The relative level of gene expression was calculated using the comparative (2 -Ct ) method. See Supplementary table 1 and table 2 for the list of primers used in the study.

Digital PCR analysis

PCRs were prepared with the required QX200 ddPCR EvaGreen Supermix (Bio-Rad) with a final concentration of 150 nM for each primer to a final volume of 20µl. Each reaction was loaded into a sample well of an 8-well disposable cartridge (Bio-Rad) followed by 70 µl of droplet generator oil (Bio-Rad), which was added to the oil wells of the cartridge. Droplets were formed in the QX200 droplet generator (Bio-Rad). Droplets were then transferred to a 96-well PCR plate, heatsealed with foil in a PX1 PCR Plate Sealer Bio-Rad and amplified with an Eppendorf Nexus Gradient master cycler (95°C primary denaturation/activation for 5min, followed by 45 cycles of 95°C for 30 sec and 61°C for 1 min, followed by 4°C 5mn and a final 98°C heat treatment for 5min). PCRs were analyzed with the QX200 droplet reader (Bio-Rad) and data analysis was performed with QuantaSoft software (version 1.7; Bio-Rad).

Supplementary

  Damian M, Marie J, Leyris JP, Fehrentz JA, Verdie P, Martinez J, Baneres JL & Mary S 2012 High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs. Journal of biological chemistry 287 3630-3641. Davis JF, Choi DL, Clegg DJ & Benoit SC 2011 Signaling through the ghrelin receptor modulates hippocampal function and meal anticipation in mice. Physiology & Behavior 103 39-43. Davis JF, Perello M, Choi DL, Magrisso IJ, Kirchner H, Pfluger PT, Tschoep M, Zigman JM & Benoit SC 2012 GOAT induced ghrelin acylation regulates hedonic feeding. Hormones and Behavior 62 598-604. Disse E, Bussier AL, Veyrat-Durebex C, Deblon N, Pfluger PT, Tschop MH, Laville M & Rohner-Jeanrenaud F 2010 Peripheral ghrelin enhances sweet taste food consumption and preference, regardless of its caloric content. Physiology & Behavior 101 277-281. Galgani J & Ravussin E 2008 Energy metabolism, fuel selection and body weight regulation. International journal of obesity 32 Suppl 7 S109-119. Ge X, Yang H, Bednarek MA, Galon-Tilleman H, Chen P, Chen M, Lichtman JS, Wang Y, Dalmas O, Yin Y, et al. 2018 LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor. Cell Metabolism 27 461-469 e466. Holst B, Cygankiewicz A, Jensen TH, Ankersen M & Schwartz TW 2003 High constitutive signaling of the ghrelin receptor--identification of a potent inverse agonist. Molecular Endocrinology 17 2201-2210. Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, et al. 1996 A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273 974-977. Inoue H, Kangawa N, Kinouchi A, Sakamoto Y, Kimura C, Horikawa R, Shigematsu Y, Itakura M, Ogata T & Fujieda K 2011 Identification and functional analysis of novel human growth hormone secretagogue Skibicka KP, Hansson C, Egecioglu E & Dickson SL 2012 Role of ghrelin in food reward: impact of ghrelin on sucrose self-administration and mesolimbic dopamine and acetylcholine receptor gene expression. Addiction Biology 17 95-107. Theander-Carrillo C, Wiedmer P, Cettour-Rose P, Nogueiras R, Perez-Tilve D, Pfluger P, Castaneda TR, Muzzin P, Schurmann A, Szanto I, et al. 2006 Ghrelin action in the brain controls adipocyte metabolism. Journal of Clinical Investigation 116 1983-1993. Tordoff MG, Alarcon LK & Lawler MP 2008 Preferences of 14 rat strains for 17 taste compounds. Physiology & Behavior 95 308-332. Torz LJ, Osborne-Lawrence S, Rodriguez J, He Z, Cornejo MP, Mustafa ER, Jin C, Petersen N, Hedegaard MA, Nybo M, et al. 2020 Metabolic insights from a GHSR-A203E mutant mouse model. Molecular metabolism 39 101004. Tschop M, Smiley DL & Heiman ML 2000 Ghrelin induces adiposity in rodents. Nature 407 908-913. Wang Q, Liu C, Uchida A, Chuang JC, Walker A, Liu T, Osborne-Lawrence S, Mason BL, Mosher C, Berglund ED, et al. 2014 Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin. Molecular metabolism 3 64-72. Zigman JM, Bouret SG & Andrews ZB 2016 Obesity Impairs the Action of the Neuroendocrine Ghrelin System. Trends Endocrinol Metab 27 54-63. Zigman JM, Jones JE, Lee CE, Saper CB & Elmquist JK 2006 Expression of ghrelin receptor mRNA in the rat and the mouse brain. Journal of Comparative Neurology 494 528-548.

FIGURE LEGENDS Figure 1 :

 LEGENDS1 FIGURE LEGENDS

Figure 2 :

 2 Figure 2: Consumption and motivation for sucrose in Ghsr M/M rats. (A) Cumulative intake of sucrose solution (0.75%) (plain lines) and water (dotted line) during daily 1h two-bottle choice in Ghsr M/M (n=17) and Ghsr WT/WT (n=13) ad libitum fed rats. (B) Operant nose-poking responses for sucrose pellets in Ghsr M/M (n=16) and Ghsr WT/WT (n=15) ad libitum fed rats during each session (upper panel) or at

Figure 3 :

 3 Figure 3: Food anticipatory activity is preserved in Ghsr M/M rats. Wheel running activity in Ghsr M/M (n=7)and Ghsr WT/WT (n=8) rats averaged over 10 days of ad libitum feeding (A) or at beginning (B) or end of the

Figure 5 :

 5 Figure 5: Increased respiratory exchange ratio in Ghsr M/M rats across feeding conditions. Daily pattern of

Figure 6 : 1 :

 61 Figure 6: mRNA expression of known markers of energy homeostasis, adiposity and glucose homeostasis in Ghsr M/M and Ghsr WT/WT rats in the hypothalamus and in peripheral tissues. qRTPCR analyses in

Table 1 .

 1 Primers sequence for real time PCR analysis

	Gene	Forward primer	Reverse primer
	name
	Acaca	GGAAAACATCCCGCACCTT	TTTCTTCTTGACCAGGTCTTCCA
	Agrp	AAGCTTTGGCAGAGGTGCTAGA	GTGCAGCCTTACACAGCGAC
	CD36	CTGTTCCTCGCCATGAAATGA	GCAGCATGGAACTTGACAACA
	Cpt1a	CTTGTTTGGCCTCACCATCA	CACGAGGGTCCGTTTTCCTT
	Fasn	TCAGAAGGCCACAGCATTCA	TTCTAAGCTGGCAAGGAGACAAC
	Ghr	GAGGCCACGACATGTGTAAAAC	GTGGTAAGGCTTTCTGTGGTGAT
	Ghsr	CCGGTCTTCTGCCTCACTGT	GCTGCATCTCCGCGTCTC
	Ghsr 1a CCTCGCTCCGGGACCAGA	AAGCAAACACCACCACAGCAA
	Ghsr 1b AGCAGACAGTGAAGATGCTTG	CTGGGGAAGGGCAAGCAGT
	Hcrt	AGAAGACGACGGCCTCAGAC	GGCAATCCGGAGAGATGGT
	Hcrtr1	GCTCTGAAGCTAGGTCCACCCT	CACAATGCCAACGAGATCCA
	Igf1	GCCGAAAATGCATGGGTG	CGGCTTCAGCAGCCAAAA
	Irs1	CAAGACGCTCCAGTGAGGATT	TTGACGGTCCTCTGGTTGCT
	Leap2	CTATGCAAAAGACGACGCTGTT	TGGAGGTGACTTCCCCAGTATG
	Lpl	CGTATCTGAGCCTGACCAAGAA	AAGGCAGGATGGTTGAGAGTTC
	Npy	CTCCGCTCTGCGACACTACA	GTGTCTCAGGGCTGGATCTCTT
	Pdk4	TGGCACTTTTACGGGATCAAA	GGAAACAAGAGTCCACACACATTC
	Pkm	AACATGCAATAGAGCCCAGCTAT	CAATCCTGCATTCCTCCTTCAC
	Pomc	TGTGAAGGTGTACCCCAATGTC	TTCCAGCTCCCTCTTGAACTCTA
	Prkaa1	CCTAACTGTAAGTCGGTTGTTGGG	TCAGGTTACTCTGGGCAAACATAC
	Slc2a1	ACATGCCTTCTTTGCCAAGCT	CATTGTTCAGTATTCGTGTGTCCTT
	Slc2a4	TCCCAAAATCTAGCCTTGCCT	GGATGTACTGTTGTGTTCCTTTGC
	Snca	AGGACCAGATGGGCAAGGG	TCCACAGGCATGTCTTCCAG
	Ucp1	TCCGGTGGATGTGGTAAAAAC	CACAGCTGGGTACACTTGGGTA
	Ucp2	CCGAAATGCCATTGTCAACTG	TGAGGTTGGCTTTCAGGAGAGTA
	Ucp3	CCTTTGTTCTACCCGTTAAGCCT	AGAAGTTGGTCCTGAACTTCGC

Table 2 .

 2 House keeping gene primers sequence for real time PCR analysis

	Gene	Forward primer	Reverse primer
	name
		Hypothalamus
	Eef1a1	ACCCTCCACTTGGTCGTTTTG	AGCTCCTGCAGCCTTCTTGTC
	Actb	CGGCAATGAGCGGTTCC	TGCCACAGGATTCCATACCC
		Liver
	Ppia	ACCCCATCTGCTCGCAATAC	GGAATGAGGAAAATATGGAACCC
	Rpl13a	TGGAGAAGAAAATCTGCAAGTTCA	TCTTTATTGGGTTCACACCAAGAGT
		WAT
	Vcp	GAGGTTTTGGCAGCTTCAGATT	ACCTCCACTGCCCTGACTTG
	Ywhaz	CTTTTTGATACTTGCCTAACATGCA	CTAGCCGTCATCTCAAGTTATTTCC
		BAT
	Sdha	TGCGGAAGCACGGAAGGAGT	CTTCTGCTGGCCCTCGATGG
	Vcp	GAGGTTTTGGCAGCTTCAGATT	ACCTCCACTGCCCTGACTTG
		Soleus-Tibialis
	Ppia	ACCCCATCTGCTCGCAATAC	GGAATGAGGAAAATATGGAACCC
	Rpl13a	TGGAGAAGAAAATCTGCAAGTTCA	TCTTTATTGGGTTCACACCAAGAGT

Acknowledgement

The authors are grateful to the PhysGen program for providing the Ghsr FHH rats (Dr. Howard Jacob,

Medical

College of Wisconsin), to the animal core and PhenoBrain facilities of the Institute of Psychiatry and Neuroscience of Paris (INSERM UMR S-1266 | Université de Paris), the animal core facility of BioMedTech (INSERM US36 | CNRS UMS2009 | Université de Paris). The authors are also thankful to Corinne Canestrelli, Claire Dovergne, Racha Fayad and Adèle Guillard for technical support, to Gabriel Montaldo for help with activity wheel recording. We also acknowledge the technical platform metabolism of the Unit "Biologie Fonctionnelle et Adaptative" (University de Paris, BFA UMR CNRS 8251) for metabolic analysis and the animal core facility "Buffon" of the University Paris Diderot Paris 7/Institut Jacques Monod, Paris for animal housing and the transcriptomic facility of INSERM U1215 Neurocenter Magendie, funded by INSERM and LabEX BRAIN ANR-10-LABX-43.

Funding

This work was supported by INSERM, by a grant of the FRM-Institut Danone (to J.P.), by the Université Sorbonne Paris Cité (USPC) for a fellowship to C.M. and by CNRS.

Supplementary materials

Supplementary figures and table (5)

Supplementary file for Material and methods (1)

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


Anaelle Humblot

Laurie Grimaud

Prince Audrey Allavena

Nana Amaniampong

Karine De Oliveira Vigier

Dr Tony Chave

Dr Stéphane Streiff

François Jerome
email: francois.jerome@univ-poitiers.fr

Prof Prince N Amaniampong

Karine De

Oliveira Vigier

François Jérôme

Karine De Oliveira

Conversion of ammonia to hydrazine induced by high frequency ultrasound

Keywords: Ammonia, Cavitation bubbles, Hydrazine, Radicals, Ultrasound

teaching and research institutions in France or abroad, or from public or private research centers.

towards a path to scavenge •OH radicals by adjusting the NH3 concentration.

Hydrazine (N2H4) is a chemical of outmost importance in the chemical industry. The hydrazine hydrate market size was 356 million USD in 2015 and is expected to reach 530 million USD in 2024, mostly boosted by the growing need of our society for the manufacture of polymer foams and agrochemicals, the actual two biggest market shares of hydrazine. [1] Besides, the rapid development of hydrazine hydrate fuel cells, [2] nowadays demonstrated with electric vehicles, should also open up new perspectives for hydrazine in the future. Projections showed that hydrazine is one of the chemicals expected to record immense growth prospects across the forecast period of 2020-2030. [3] Hydrazine is industrially produced by partial oxidation of NH3, either with hypochlorite or with peroxide. [4] In the widest spread industrial processes (Bayer-ketazine and peroxide process), hydrazine is trapped in situ under the form of a ketazine, by reaction with a ketone, to prevent its decomposition (Fig. S1). Ketazine is then hydrolyzed to release hydrazine and regenerate the ketone. The direct production of hydrazine from NH3 or N2/H2O is economically and environmentally highly attractive, but it remains a very difficult task. One of the reason stems from the high N-H and N-N bond dissociation of NH3 (435 kJ/mol) and N2 (946 kJ/mol), respectively, requiring harsh conditions of temperature and pressure, which are not compatible with the stability of hydrazine. Indeed, the decomposition of hydrazine is thermodynamically more favorable (G = -150 kJ/mol) than the conversion of NH3 (G = 16.5 kJ/mol) or N2/H2O (G = 184 kJ/mol) to hydrazine, making the accumulation of hydrazine scientifically challenging. [5] Physical activation of the N-H and N-N bond of NH3 and N2, respectively, at low temperature with electron beam, [6] plasma, [7] light, [8] or by the coupling of these technologies with catalysis such as electrocatalysis for instance have been explored for the synthesis of hydrazine. [5a, 9] Although fascinating results were reported, the in situ decomposition of hydrazine cannot be avoided, leading to the formation of very diluted feeds of hydrazine (i.e. micromolar). High frequency ultrasound (HFUS), a technology based on cavitation phenomenon, is gaining more and more interest in the field of chemistry. [START_REF]Sonochemistry: from Basic Principles to Innovative Applications[END_REF] Due to the extreme conditions of pressures and temperatures existing inside the cavitation bubbles, it was shown by sonoluminescence spectroscopy that gaseous molecules, with high bond dissociation energies such as N2, O2, CO2, CH4, can be cleaved inside the cavitation bubbles, leading to the formation of radicals and the occurrence of sonochemical reactions. [11] So far, the activation of NH3 by HFUS has been investigated sporadically, and the rare previous studies were mainly focused on the elimination of NH3 from waste water (by degassing, pyrolysis). [12] In 2017, a pioneer work of Pflieger demonstrated, by sonoluminescence spectroscopy, that NH species can be formed inside the cavitation bubbles, by subjecting an aqueous solution of NH3 (0,17 wt%) to an ultrasonic irradiation at 359 kHz. [13] Inspired by these results, we investigate here the sonochemical conversion of NH3 to N2H4. Unlike previous works, we show here that by adjusting the temperature of the solution and the concentration of NH3, it was possible to limit the decomposition of hydrazine, a scientific and technological problem faced by previous routes. In a first set of experiments, NH3 was continuously bubbled (30 mL/min) in 100 mL of water subjected to an ultrasonic irradiation at 525 kHz, with an acoustic power of 0.17 W/mL. The aqueous solution of NH3 was maintained at 30°C using a cooling system coupled to the ultrasonic reactor (Fig. S2). Hydrazine was titrated by spectrophotometry and results were doubly confirmed by HPLC titration (Fig. S3-S7). Hereafter, the formation rate of hydrazine is given with an uncertainty of 5%. Pleasingly, under these conditions, hydrazine was continuously produced at a rate of 0.12 mmol.L -1 .h -1 (Fig. S8). The dilution of NH3 in gases such as O2, N2, Ar, or He resulted in a decrease of the formation rate of hydrazine (Fig. 1). No correlation was established between the physicochemical properties of the gas (thermal conductivity, heat capacity, etc…) and the formation rate of N2H4. As a result, we suspect that the formation rate of N2H4 is more related to the solubility of NH3 in water. To support this claim, aqueous NH3 solutions with different concentrations (from 0.1 to 20 wt%) were subjected to an ultrasonic irradiation (525 kHz) under air (i.e. no bubbling of NH3). The plot of the formation rate of hydrazine as a function of the NH3 concentration is reported in Fig. 2. A volcano-type curve was obtained. The formation rate of hydrazine gradually increased with a concomitant increase of the NH3 concentration from 0.1 to 7 wt%, to reach a maximum of 0.17 mmol N2H4.L -1 .h -1 at 7 wt% NH3. At higher concentration of NH3, the formation rate of hydrazine declined, and was even inhibited at a NH3 concentration over 15 wt%. This result can be understood in terms of thermodynamics. The sonochemical decomposition of NH3 is endothermic. Hence, when the concentration of NH3 inside the bubbles is increasing, it decreases the maximum temperature and pressure that can be reached inside the cavitation bubbles, thus finally quenching sonochemical reactions. [14] In order to get more insight on the reaction mechanism, we first checked any possible role of the metallic reactor wall (316 L stainless steel) on the hydrazine production. To this end, a similar experiment was conducted by confining the aqueous solution of NH3 (5 wt%) in a polyethylene flask (material transparent to the ultrasonic waves) immerged into the ultrasonic reactor filled with water. No significant change in hydrazine production rate was observed, ruling out a possible contribution of the metallic reactor wall in the reaction mechanism (Fig. S9-S10). Next, the concentration of hydrazine was plotted as a function of the reaction time (30°C, NH3 at 30 mL/min, 0.21 W/mL). Very interestingly, over a period of 20 h, the reaction never plateaued and hydrazine was continuously produced (Fig. 3). At 20 h of ultrasonic irradiation at 525 kHz, the concentration of hydrazine reached 3.3 mmol/L, which is about 1000 times higher than in previously reported works.

These results also suggest that the formation rate of hydrazine is faster than its decomposition, which constitute one of the unique examples in the state of the art. One of the explanations for this result stems from the low temperature of the aqueous NH3 solution (30°C), preventing the thermal decomposition of hydrazine. Indeed, under silent conditions, hydrazine was found stable in aqueous NH3 (5 wt%) at 30°C, while at 60°C a degradation was observed (Fig. S11).

To support this claim, the temperature of the ultrasonic reactor was increased from 30 to 60°C. At 60°C, the formation of hydrazine remained very low and reached only 0.073 mmol/L after 60 min, (Fig. 3), confirming the negative impact of the temperature on the hydrazine production. Although high temperatures are reached in the cavitation bubbles, the bulk aqueous solution of NH3 can be maintained at 30°C, which represents a noticeable advantage of this technology to prevent the thermal degradation of hydrazine.

The second explanation can be understood in terms of •OH radical scavenging by NH3. It has been previously demonstrated, by sonoluminescence spectroscopy, that ultrasonic irradiation of an aqueous solution of NH3 concomitantly generate NH and •OH radicals. [13] •OH radicals are known to decompose hydrazine. [15] To assess the reactivity of in situ produced •OH radicals, we first measured the amount of H2O2 formed, resulting from the recombination of •OH radicals (Fig. S12, 13). Interestingly, when the concentration of NH3 is over 1 wt%, no H2O2 was detected, confirming that •OH radicals are quickly scavenged in situ (Fig. •OH radicals by NH3, to form •NH2 radical and water, or (2) the scavenging of •OH radicals by hydrazine, inducing its partial decomposition to N2 and H2O. To discriminate between these two scenarios, a solution of hydrazine (0.1 mmol/L) was subjected to an ultrasonic irradiation either with or without NH3.

In line with previous reports, without NH3, hydrazine was nearly completely decomposed after only 2 h of irradiation by in situ produced •OH radicals (Fig. 4). In contrast, in an aqueous solution of NH3 (5 wt%), the concentration of hydrazine kept increasing, indicating that •OH radicals are scavenged by NH3 (Fig. 4). A similar behavior was observed with 1 mmol/L of hydrazine (Fig. S15). These results can be explained in terms of probability. Indeed, the concentration of NH3 (1-5 wt% = 0.6-3 mol/L) is much higher than that of hydrazine. As a result, the probability for the •OH radical to react with NH3 is much higher than with hydrazine. From these data, a plausible reaction mechanism can be proposed. During the ultrasonic irradiation, NH3 and vapors of water diffuse within the cavitation bubbles to produce NH and

•OH radicals as observed elsewhere. [13] NH species can react either with NH3 to form two •NH2 radicals or with H2O to form •NH2 and •OH radicals. These recombination reactions can occur either inside the cavitation bubbles or at the cavitation bubble-liquid interface. As the amount of H2O is 18 times higher than that of NH3 in our reactor (at 5 wt% NH3), we assume that the main reaction taking place for NH is the reaction with H2O.

On the other hand, and as discussed above, •OH radicals are rapidly trapped by NH3, the second largest abundant chemical in the HFUS reactor, presumably to form •NH2 radicals and water, a reaction which is thermodynamically favored (G = -46 kJ/mol). [16] In terms of probability, the recombination of •NH2 radicals with •OH radicals is less likely to occur, as •OH radicals are rapidly scavenged by NH3. To check this hypothesis, the asobtained aqueous solution was fully analyzed by HPLC, in particular to detect the formation of nitrate. Indeed, it is known that •OH radical can react with •NH2 radical to form NH2OH which is instable and further rapidly convert to nitrate. [17] As expected, with a bubbling of NH3 (30 mL/min), the amount of nitrate remained below the detection limit of our apparatus, supporting that the recombination of •NH2 with •OH is not a dominant reaction. Hence, •NH2 radicals has no other option than to dimerize to form N2H4.

In an attempt to elucidate whether hydrazine is formed inside the cavitation bubbles or at the bubble liquid interface, phenol was added into the solution as a radical scavenger. Phenol has a high boiling point (182°C) and a low vapor pressure (0.2 mmHg at 20°C), making it improbable its diffusion inside the cavitation bubble, as demonstrated in other reports. [18] Hence, phenol is a suitable radical scavenger to selectively trap radicals at the bubble-liquid interface. Interestingly, in the presence of phenol (0.1 mol/L) in an aqueous NH3 solution (5 wt%), no hydrazine was formed, suggesting that hydrazine is more likely to be formed at the bubble-liquid interface. To further support this claim, sodium benzoate was also tested as another radical scavenger, as it reacts slower with radical species than phenol, allowing a better monitoring of the reaction. [16,19] In addition, as a salt, the vapor pressure of sodium benzoate is negligible at 30°C. As expected, an incremental increase of the amount of sodium benzoate led also a gradual decrease of the amount of hydrazine, confirming the results obtained with phenol (Fig. S16).

Altogether, these results strongly suggest that the formation of hydrazine occurred at the bubble-liquid interface. Beside, hydrazine hydrate (64 wt%) has a boiling point of 114°C and a vapor pressure of 7.5 mmHg at 30°C. Hence, its diffusion inside the cavitation bubbles is negligible and thus hydrazine dominantly remains compartmented into the bulk solution at 30°C, where its thermal decomposition is drastically limited. (Fig. 5). aqueous NH3 solution, respectively, which are typical concentrations routinely handled in industry. The highest amount of N2H4 is obtained at the highest NH/OH ratio (Fig. S20). It is noteworthy that at these concentration ranges, and in our conditions (atmospheric pressure and 30°C), the risk of detonation/flammability is very unlikely (no flash point of aqueous hydrazine at these concentrations), water acting as an inerting agent in this case. [20] More information on safety is discussed in the supporting information.

With the reactor design in our hands, we are limited by the low formation rate of N2H4, making it very long, and even not realistic, the formation of 2-8 wt% aqueous solution of hydrazine. However, the formation rate of hydrazine could be largely increased by working in a continuous flow reactor, as the acoustic power density will be locally much higher than in our reactor. To demonstrate this possibility, the acoustic power density was varied from 0.1 to 0.23 W/mL (Fig. 6). As expected, the formation rate of hydrazine was directly impacted by the acoustic power and the formation rate of hydrazine exponentially increased with the acoustic power. This last result demonstrates that switching from a batch to a continuous flow reactor could be a promising perspective for this work to investigate the conversion of NH3 to more concentrated solutions of hydrazine hydrate, but also to reduce the energy expenditure of this pathway.

Figure 6. Effect of the absorbed acoustic power on the formation rate of hydrazine (30 ml/min NH3, 525 kHz, 30°C).

In conclusion, these preliminary results suggest that high ultrasound is a potential disruptive technology for the direct, salt-free conversion of NH3 to hydrazine. Cavitation bubbles act as micro-reactors to activate NH3, resulting in the formation of NH species that are further recombined to hydrazine at the bubble-liquid interface. One of the advantage of this technology is the compartmentation of hydrazine into the bulk solution where the temperature is maintained at 30°C, thus avoiding its thermal degradation, a recurrent problem faced with previous technologies. In addition, we showed that adjusting the concentration of NH3 allowed a rapid scavenging of •OH by NH3, thus limiting the oxidative degradation of hydrazine at the bubble liquid-interface. Two perspectives are under investigation in our groups (1) a switch from a batch to a continuous flow reactor to improve the efficiency of this transformation, in particular in terms of reaction time (i.e. energy efficiency), and (2) the exploration of this technology for the activation and utilization of NH3 in organic reactions to produce chemicals with higher added value than N2H4. Acoustic power (W/mL)

Entry for the Table of Contents

Cavitation bubbles act as micro-reactors to activate NH3, resulting in the formation of NH species that are further recombined to hydrazine at the bubble-liquid interface. One of the advantages of this technology is the compartmentation of the as-formed hydrazine in the bulk solution, maintained at only 30°C, which limits its thermal degradation.

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Figure 2 .

 2 Figure 2. Effect of the NH3 concentration on the formation rate of hydrazine (525 kHz, 0.17 W/mL, 30°C).

Figure 3 .

 3 Figure 3. Concentration of hydrazine as a function of the reaction time (bubbling of NH3 at 30 ml/min, 525 kHz, 0.21 W/mL) at 30°C and 60°C.

  scenarios are thus possible: (1) the scavenging of

Figure 4 .

 4 Figure 4. Stability of hydrazine under ultrasonic irradiation with and without NH3 (initial hydrazine concentration = 0.1 mmol/L in water or in 5 wt% ammonia, 525 kHz, 30°C, 0.17 W/mL).

Figure 5 .

 5 Figure 5. Proposed reaction pathway. For the sake of clarity, only reactions taking place at the cavitation bubble-liquid interface are shown.

Acknowledgements

Authors are grateful to the CNRS, the University of Poitiers and the region Nouvelle Aquitaine for the funding of this study. AH is also grateful to the CNRS for the funding of her PhD. Authors also acknowledge Marie Jérôme for the statistic calculations. YingYing Ma, Bright Kusema (Health Safety and Environment managers from SOLVAY) and emeritus Prof Charles Kappenstein are also acknowledged for the discussion on the safety of hydrazine.