Antonela S Fittipaldi

Julieta Hernández

Daniel Castrogiovanni

Daniela Lufrano

Pablo N De Francesco

Verónica Garrido

María Patrick Vitaux

María Victoria Fasano

Jean-Alain Fehrentz

Adriana Fernández

María F Andreoli
email: mfandreoli@gmail.com

Mario Perello
email: marioperello@yahoo.com

Plasma levels of ghrelin, des-acyl ghrelin and LEAP2 in children

à la diffusion 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.

Introduction

Ghrelin is a gastrointestinal tract-derived hormone that acts via the growth hormone secretagogue receptor (GHSR) [START_REF] Kojima | Ghrelin is a growth-hormone-releasing acylated peptide from stomach[END_REF]. Ghrelin is a 28-residue peptide that contains an n-octanoyl ester at its third serine residue, an unusual posttranslational modification catalyzed by the enzyme ghrelin O-acyltransferase [START_REF] Yang | Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone[END_REF]. This lipid modification is essential for the bioactivity of ghrelin [START_REF] Kojima | Ghrelin is a growth-hormone-releasing acylated peptide from stomach[END_REF]. Administration of ghrelin to healthy individuals increases hunger/food intake and promotes mechanisms that increase glycaemia [START_REF] Akamizu | Pharmacokinetics, safety, and endocrine and appetite effects of ghrelin administration in young healthy subjects[END_REF]. Administration of ghrelin decreases insulin secretion [START_REF] Tong | Ghrelin suppresses glucose-stimulated insulin secretion and deteriorates glucose tolerance in healthy humans[END_REF] and increases plasma levels of growth hormone (GH), adrenocorticotropic hormone and cortisol [START_REF] Akamizu | Pharmacokinetics, safety, and endocrine and appetite effects of ghrelin administration in young healthy subjects[END_REF]. In plasma, ghrelin also exists as a des-octanoylated form, hereafter named des-acyl ghrelin (DAG). DAG is either secreted from ghrelin-producing cells of the gastrointestinal tract or results from ghrelin des-acylation in plasma [START_REF] Yang | Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone[END_REF]. The role of DAG is uncertain since DAG does not bind to GHSR at physiological ranges, and no DAG receptor has been identified, yet. Strikingly, DAG modulates genes involved in glucose and lipid metabolism in mice lacking GHSR, suggesting a GHSR-independent action [START_REF] Delhanty | Unacylated ghrelin rapidly modulates lipogenic and insulin signaling pathway gene expression in metabolically active tissues of GHSR deleted mice[END_REF]. In humans, plasma DAG levels are higher than ghrelin levels, and some studies show that DAG infusion improves glucose metabolism [START_REF] Benso | Metabolic effects of overnight continuous infusion of unacylated ghrelin in humans[END_REF][START_REF] Ozcan | Does des-acyl ghrelin improve glycemic control in obese diabetic subjects by decreasing acylated ghrelin levels?[END_REF] and that concomitant administration of DAG and ghrelin abolishes the hyperglycaemic effects of ghrelin [START_REF] Broglio | Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans[END_REF]. However, other studies could not confirm such observations [START_REF] Tong | Acute administration of unacylated ghrelin has no effect on Basal or stimulated insulin secretion in healthy humans[END_REF]. Recently, liver antimicrobial peptide 2 (LEAP2) has been identified as another ligand of GHSR [START_REF] Ge | LEAP2 is an endogenous antagonist of the ghrelin receptor[END_REF]. LEAP2 is a 40-residue peptide primarily secreted by the liver and jejunum [START_REF] Ge | LEAP2 is an endogenous antagonist of the ghrelin receptor[END_REF]. LEAP2 blocks ghrelin-induced activation of GHSR and also reduces GHSR constitutive activity [START_REF] Kadmi | N-terminal liver-expressed antimicrobial Peptide 2 (LEAP2) region exhibits inverse agonist activity toward the ghrelin receptor[END_REF][START_REF] Wang | Identifying the binding mechanism of LEAP2 to receptor GHSR1a[END_REF]. In mice, LEAP2 blocks the stimulatory effects of ghrelin on food intake and GH secretion, and plasma LEAP2 levels display an inverse relationship to plasma ghrelin levels: LEAP2 levels decrease under fasting and increase to basal levels upon refeeding [START_REF] Ge | LEAP2 is an endogenous antagonist of the ghrelin receptor[END_REF].

The accurate assessment of ghrelin in plasma has been proved to be challenging. Initial studies investigating plasma ghrelin levels in human samples reported ghrelin plus DAG levels (usually referred to as total ghrelin), as the early available commercial immunoassays did not discriminate the acylated and desacylated forms of the peptide [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Hosoda | Standard sample collections for blood ghrelin measurements[END_REF]. Only the subsequent development of immunoassays to specifically assess ghrelin or DAG allowed the independent quantification of these two peptides in plasma. The assessment of ghrelin is also affected by the instability of the octanoyl modification since the ester bond is highly susceptible to be spontaneously hydrolysed at physiological pH and also a substrate of plasma esterases that rapidly des-acylate ghrelin [START_REF] Delhanty | The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor-treated blood is higher than previously described[END_REF]. Only recently the methods of collection, handling and storage of plasma samples have been standardized for the correct assessment of ghrelin [START_REF] Hosoda | Standard sample collections for blood ghrelin measurements[END_REF].

Different studies show an association between ghrelin and obesity. In adults, ghrelin levels decrease in patients with obesity and negatively correlate with BMI [START_REF] Shiiya | Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion[END_REF][START_REF] Tschop | Circulating ghrelin levels are decreased in human obesity[END_REF]. Ghrelin levels also decrease in children and adolescents with obesity [START_REF] Reinehr | Obestatin and ghrelin levels in obese children and adolescents before and after reduction of overweight[END_REF] and negatively correlate with BMI [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Soriano-Guillen | Effect of oral glucose administration on ghrelin levels in obese children[END_REF][START_REF] Whatmore | Ghrelin concentrations in healthy children and adolescents[END_REF]. Notably, only few studies have independently assessed ghrelin and DAG levels in children with obesity, and observations have been inconsistent [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Mackelvie | Regulation of appetite in lean and obese adolescents after exercise: role of acylated and desacyl ghrelin[END_REF][START_REF] Pacifico | Relationships of acylated and des-acyl ghrelin levels to bone mineralization in obese children and adolescents[END_REF][START_REF] Razzaghy-Azar | An evaluation of acylated ghrelin and obestatin levels in childhood obesity and their association with insulin resistance, metabolic syndrome, and oxidative stress[END_REF]. Many studies found that ghrelin levels negatively correlate with circulating insulin and insulin resistance in adults and children [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Razzaghy-Azar | An evaluation of acylated ghrelin and obestatin levels in childhood obesity and their association with insulin resistance, metabolic syndrome, and oxidative stress[END_REF][START_REF] Cheng | Ghrelin and peptide YY change during puberty: relationships with adolescent growth, development, and obesity[END_REF][START_REF] Ikezaki | Fasting plasma ghrelin levels are negatively correlated with insulin resistance and PAI-1, but not with leptin, in obese children and adolescents[END_REF][START_REF] Soriano-Guillen | Sex-related differences in the association of ghrelin levels with obesity in adolescents[END_REF]. In contrast, studies looking for associations between plasma ghrelin and circulating lipids have reported inconsistent results [START_REF] Razzaghy-Azar | An evaluation of acylated ghrelin and obestatin levels in childhood obesity and their association with insulin resistance, metabolic syndrome, and oxidative stress[END_REF][START_REF] Park | Relationships between fasting plasma ghrelin levels and metabolic parameters in children and adolescents[END_REF][START_REF] Purnell | Ghrelin levels correlate with insulin levels, insulin resistance, and high-density lipoprotein cholesterol, but not with gender, menopausal status, or cortisol levels in humans[END_REF][START_REF] Zou | Factors associated with fasting plasma ghrelin levels in children and adolescents[END_REF][START_REF] Wadden | Serum acylated ghrelin concentrations in response to short-term overfeeding in normal weight, overweight, and obese men[END_REF]. Thus, it seems evident that more clinical studies performing careful biochemical analyses in diverse human populations are still necessary for a better understanding of the implications of the ghrelin system in human beings. Regarding LEAP2, a recent study reports that LEAP2 levels are higher in patients with morbid obesity and positively correlate with BMI and plasma glucose [START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF]. To our knowledge, plasma LEAP2 levels have not been investigated in children.

As a manoeuvre to assess the status of the ghrelin system, plasma levels of ghrelin and DAG have been combined to generate several indices. The sum of ghrelin plus DAG could be considered indicative of the total ghrelin production. Since DAG appears to be a functional inhibitor of ghrelin [START_REF] Ozcan | Does des-acyl ghrelin improve glycemic control in obese diabetic subjects by decreasing acylated ghrelin levels?[END_REF][START_REF] Delhanty | The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor-treated blood is higher than previously described[END_REF], the ghrelin/DAG ratio has been used to estimate the effective ghrelin tone. Some studies show that the ghrelin/DAG ratio is a useful biomarker of excessive weight gain, even prior to the start of hyperphagia in subjects with Prader-Willi syndrome [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF]. Increased ghrelin/DAG ratio is also linked to obesity and diabetes [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Barazzoni | Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome[END_REF][START_REF] Inhoff | Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats[END_REF][START_REF] Vivenza | Ghrelin gene polymorphisms and ghrelin, insulin, IGF-I, leptin and anthropometric data in children and adolescents[END_REF]. Since LEAP2 blocks ghrelin actions, we hypothesized that the ghrelin/LEAP2 ratio could provide another estimation of the effective ghrelin tone. The aim of our study was to evaluate the plasma levels of ghrelin, DAG and LEAP2 in children with overweight or obesity and their association with different anthropometric and metabolic variables, investigating also a potential sexual dimorphism.

Methods

Study design

The study was approved by the Institutional Committee for the [START_REF] De Onis | Development of a WHO growth reference for school-aged children and adolescents[END_REF].

As described by the WHO, children with a BAZ between -2 and +1 were considered normoweight (NW), above +1 BAZ were considered patients with overweight (OW), and above +2 BAZ were considered patients with obesity (OB) [START_REF] De Onis | Development of a WHO growth reference for school-aged children and adolescents[END_REF]. Children with a BAZ lower than -2 were considered wasted and were not included in this study.

Blood samples

The participants were asked not to eat after 21:00 h the night before the session. Between 09:00 and 11:00 h, an overnight fasting blood sample was drawn from each child and collected on EDTA (1 mg/mL final) to measure plasma levels of glucose, insulin, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C) and triacylglycerols (TG) levels. For ghrelin, DAG and LEAP2 determinations, blood samples were collected on EDTA (1 mg/mL final) and p-hydroxy-mercuribenzoic acid (0.4 mM final) and, then, plasmas were immediately acidified with HCl (0.1 N final) to preserve acylation [START_REF] Hosoda | Standard sample collections for blood ghrelin measurements[END_REF]. All samples were stored frozen at -80°C until analysis.

Laboratory analyses

Glucose, TC, TG, LDL-C, VLDL-C and HDL-C levels were measured using standard enzymatic procedures in a TARGA BT3000 PLUS analyser (Biotecnica Instruments, Italy). Insulin levels were assessed by chemiluminescence (Access Beckman Coulter, Switzerland). Insulin resistance was estimated by the homeostasis model assessment for insulin resistance (HOMA index) [START_REF] Matthews | Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man[END_REF]. Ghrelin and DAG levels were assessed using specific enzyme immunoassays (A05306 and A05319, respectively, Bertin Bioreagent, Bertin Technologies, France). Assays were performed according to manufacturer's instructions. Briefly, 100 µL of standards, quality controls and samples (diluted 1:2 or 1:5 in buffer for ghrelin or DAG determinations, respectively) were added to the plate as well as 100 µL of the tracer antibody and incubated overnight at 4°C. Following three washes, Ellman's reagent was added. After 60 min, the absorbance was measured at 405 nm using a Packard SpectraCount BS10000 absorbance microplate reader (USA). LEAP2 levels were assessed using a specific enzyme immunoassay for human and mouse LEAP2 detection (EK-075-40, Phoenix Pharmaceutical, USA) according to manufacturer's instructions. This assay demonstrated linearity of dilution and parallelism and detected lower levels of LEAP2 in plasma samples from calorie-restricted mice [START_REF] Cornejo | Growth hormone secretagogue receptor signalling affects high-fat intake independently of plasma levels of ghrelin and LEAP2, in a 4-day binge eating model[END_REF]. This LEAP2 ELISA assay kit has been recently validated in human samples [START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF]. Briefly, 25 µL of standards, quality controls and samples (diluted 1:25 in buffer) were incubated in the plate at room temperature with 50 µL of antibody. After 2 h, 25 µL of biotinylated LEAP2 were added and incubated overnight at 4°C. Following several washes, horseradish peroxidase was added and incubated for 1 h. Following three washes, 3,3′,5,5′-tetramethylbenzidine was added and incubated for 1 h. Finally, the reaction was stopped with HCl and absorbance was measured at 405 nm as described earlier.

Ghrelin, DAG and LEAP2 levels were calculated from the corresponding calibration curves using logarithmic fitting in GraphPad Prism 5. Ghrelin + DAG levels were computed on a molar basis by adding ghrelin and DAG concentrations. Ghrelin/DAG ratio was computed on a molar basis as ghrelin concentration divided by DAG concentration. Ghrelin/LEAP2 ratio was computed on a molar basis as ghrelin concentration divided by LEAP2 concentration. The interaction term gender*BAZ was included in the ANOVA model and if it was considered not significant, the ANOVA was run again only with the main effects. Distribution of residuals was checked using the Shapiro-Wilk test and normal quantile plots. The statistical tests were two tailed, and the significance level was set at 5%. In order to study interdependencies among ghrelin, DAG, LEAP2 and variables that showed significant correlation with any other variable (BAZ, insulin, TG, VLDL-C, LDL-C and HDL-C), a hierarchical clustering was performed. This data set was normalized with mean = 0 and s.d. = 1, and data from children with NW and children with OW/OB were processed separately to construct their corresponding ordered heatmaps with dendrograms for participants and variables. The dendrograms were computed by agglomerative hierarchical clustering, using Euclidean distance metrics and group average as the similarity criterion. This analysis was performed using Orange software [START_REF] Demsar | Orange: data mining toolbox in Python[END_REF].

PROOF ONLY

Results

Characteristics of study subjects

Among the 82 subjects, 36 (44%) and 46 (66%) children met BAZ criteria for NW or OW/OB, respectively. The OW/OB group included 18 children with OW and 28 children with OB. Among children with OB, 21 children had a BAZ between +2 and +4, and 7 children had a BAZ higher than +4. The study included 42 (51%) females and 40 (49%) males. Table 1 shows some characteristics of the participant children. The age and birth weight were similar between groups, while WAZ and BAZ were higher in children with OW/OB, as compared to children with NW, as expected. Also, insulin levels and HOMA index were higher and HDL-C was lower in children with OW/ OB, as compared to children with NW. The differences in HAZ between both groups, although statistically significant, are between the normal ranges. No changes were observed in other variables.

Plasma levels of ghrelin, DAG and LEAP2

Table 2 shows plasma levels of ghrelin, DAG and LEAP2 as well as indices based on these variables for children with NW or OW/OB, in all subjects and in male and female children separately. Ghrelin levels were similar between groups, while levels of DAG, ghrelin + DAG and LEAP2 were lower in children with OW/OB, as compared to children with NW, independently of the gender. Ghrelin/DAG ratio was higher in children with OW/OB, as compared to children with NW, independently of the gender. Ghrelin/LEAP2 ratio did not differ between groups. As the interaction term gender*BAZ was not significant in any comparison, it was excluded from the ANOVA.

Correlation analyses

First, variables shown in Table 1 were subjected to correlation analyses with variables shown in Table 2.

Results of correlation analyses are shown in Table 3. When all subjects were analysed together, ghrelin levels did not correlate with any assessed variable. In contrast, DAG, LEAP2 and ghrelin + DAG levels as well as the ratio of ghrelin/DAG significantly correlated with different variables. In particular, DAG levels negatively correlated with age, BAZ, glycaemia (r = -0.24, P = 0.0307), insulin levels and HOMA index. Ghrelin + DAG levels positively correlated with age, BAZ, insulin levels and HOMA index, but did not correlate with glycaemia. LEAP2 levels negatively correlated with BAZ and birth weight (r = -0.27, P = 0.0147). Ghrelin/DAG ratio positively correlated with age, BAZ, insulin levels and HOMA index, while ghrelin/ LEAP2 ratio did not correlate with any assessed variable. For the lipid panel, no significant correlations were found with the exception of LEAP2 levels that negatively correlated only with LDL-C levels. No significant correlations were found between ghrelin, DAG, LEAP2 or their indices and HAZ in any case.

Next, the same correlation analyses described earlier were separately conducted in children with either NW or OW/OB (Table 3). Ghrelin levels did not correlate with any assessed variable in any of the groups. DAG levels negatively correlated with age, insulin levels and HOMA index in both groups of children, and the correlations were stronger in children with OW/OB vs NW. DAG levels negatively correlated with glycaemia only in children with NW (r = -0.39, P = 0.0199), while it negatively correlated with BAZ and positively correlated with HDL only in children with OW/OB. Ghrelin + DAG levels negatively correlated with age in both groups of children. Ghrelin + DAG levels negatively correlated with BAZ, insulin levels and HOMA index and positively correlated with HDL levels only in children with OW/OB. LEAP2 levels negatively correlated with BAZ only in children with NW, while it negatively correlated with birth weight (r = -0.37, P = 0.0122) only in children with OW/OB. Ghrelin/DAG ratio positively correlated with age in both groups of children and with insulin levels and HOMA index only in children with OW/OB. The ghrelin/LEAP2 ratio did not correlate with any assessed variable in any group. For the lipid panel, no significant correlations were found with the exception of LEAP2 levels that negatively Finally, correlation analyses of the same variables described earlier were separately conducted in male and female children (Table 4). Ghrelin levels did not correlate with any assessed variable in male or female children. DAG and ghrelin + DAG levels negatively correlated with age, BAZ, insulin levels and HOMA index in male and female children. Strikingly, DAG levels positively correlated with LDL levels only in male children and negatively correlated with TG, LDL-C and VLDL-C levels only in female children. Ghrelin + DAG levels negatively correlated with TG and VLDL-C levels only in female children. LEAP2 levels negatively correlated with BAZ only in male children, while it negatively correlated with birth weight (r = -0.38, P = 0.0162) only in female children. Ghrelin/DAG ratio positively correlated with age, insulin levels and HOMA index in both male and female children. In male children, ghrelin/DAG ratio negatively correlated with LDL-C levels and positively correlated with BAZ, while in female children, it negatively correlated with TG, LDL-C and VLDL-C levels. The ghrelin/LEAP2 ratio did not correlate with any assessed variable in any group.

Study of interdependencies by agglomerative hierarchical clustering

Hierarchical clustering showed that metabolic and anthropometric variables cluster differently in children with NW or with OW/OB (Fig. 1). In NW, ghrelin and LEAP2 clustered together as well as LDL and DAG. In children with OW/OB, ghrelin and LEAP2 are no longer clustered together, while DAG clusters with HDL.

Discussion

Here, we assessed the plasma levels of ghrelin, DAG and LEAP2 in children with NW or with OW/OB and investigated their association with anthropometric and metabolic variables. To our knowledge, this is the first clinical study that reports circulating levels of LEAP2, a recently discovered GHSR ligand, in a paediatric population. In addition, we explored the association of ghrelin/DAG and ghrelin/LEAP2 ratios with different anthropometric and metabolic variables.

The paediatric population under study included 46 children that met BAZ criteria for OW/OB. We found high plasma insulin levels and HOMA index in children with OW/OB, indicating insulin resistance. Also, we found that HDL-C was 8% lower, while total cholesterol, LDL-C and VLDL-C remained unchanged, showing that alterations in glucose homeostasis appear early in children with OW/OB, and they are more evident than changes in the lipid profile. These outcomes agree with the notion that decreased insulin sensitivity and dyslipidaemia can occur in children and adolescents as a result of obesity [START_REF] Daniels | Complications of obesity in children and adolescents[END_REF]. Also, we found lower DAG levels, a slight (8%) but not significant reduction of ghrelin levels, and a consequent reduction of the ghrelin/DAG ratio and the ghrelin + DAG levels in children with OW/OB. To the best of our knowledge, ghrelin and/or DAG levels in children were independently assessed only in six studies [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Mackelvie | Regulation of appetite in lean and obese adolescents after exercise: role of acylated and desacyl ghrelin[END_REF][START_REF] Pacifico | Relationships of acylated and des-acyl ghrelin levels to bone mineralization in obese children and adolescents[END_REF][START_REF] Razzaghy-Azar | An evaluation of acylated ghrelin and obestatin levels in childhood obesity and their association with insulin resistance, metabolic syndrome, and oxidative stress[END_REF][START_REF] Vivenza | Ghrelin gene polymorphisms and ghrelin, insulin, IGF-I, leptin and anthropometric data in children and adolescents[END_REF]. A reduction of DAG levels has been consistently reported in adults [START_REF] Barazzoni | Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome[END_REF][START_REF] Marzullo | The relationship between active ghrelin levels and human obesity involves alterations in resting energy expenditure[END_REF], adolescents [START_REF] Mackelvie | Regulation of appetite in lean and obese adolescents after exercise: role of acylated and desacyl ghrelin[END_REF][START_REF] Pacifico | Relationships of acylated and des-acyl ghrelin levels to bone mineralization in obese children and adolescents[END_REF] and pre-pubertal children [START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Pacifico | Relationships of acylated and des-acyl ghrelin levels to bone mineralization in obese children and adolescents[END_REF] with obesity. In contrast, studies assessing ghrelin levels in individuals with obesity reported inconsistent results. Some studies found a significant decrease (33-45%) of ghrelin levels in adults [START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF][START_REF] Marzullo | The relationship between active ghrelin levels and human obesity involves alterations in resting energy expenditure[END_REF][START_REF] Tentolouris | Differential effects of high-fat and highcarbohydrate content isoenergetic meals on plasma active ghrelin concentrations in lean and obese women[END_REF] and pre-pubertal [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Razzaghy-Azar | An evaluation of acylated ghrelin and obestatin levels in childhood obesity and their association with insulin resistance, metabolic syndrome, and oxidative stress[END_REF] and pubertal children [START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF] with obesity. In contrast, and in agreement with our results, other studies reported a slight (16%) or no reduction of ghrelin levels in adults [START_REF] Wadden | Serum acylated ghrelin concentrations in response to short-term overfeeding in normal weight, overweight, and obese men[END_REF][START_REF] Barazzoni | Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome[END_REF], children and adolescents [START_REF] Mackelvie | Regulation of appetite in lean and obese adolescents after exercise: role of acylated and desacyl ghrelin[END_REF][START_REF] Pacifico | Relationships of acylated and des-acyl ghrelin levels to bone mineralization in obese children and adolescents[END_REF][START_REF] Vivenza | Ghrelin gene polymorphisms and ghrelin, insulin, IGF-I, leptin and anthropometric data in children and adolescents[END_REF] with obesity. The reasons for the divergent findings are unknown, but may be related to methodological differences in plasma sampling and storage, which are critical for a hormone inherently unstable such as ghrelin. Importantly, we confirmed that ghrelin + DAG levels are decreased in individuals with OW/OB, as reported in most studies [START_REF] Shiiya | Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion[END_REF][START_REF] Tschop | Circulating ghrelin levels are decreased in human obesity[END_REF][START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Purnell | Ghrelin levels correlate with insulin levels, insulin resistance, and high-density lipoprotein cholesterol, but not with gender, menopausal status, or cortisol levels in humans[END_REF]. Since ghrelin is converted to DAG in plasma, the simultaneous assessment of ghrelin and DAG appears as a better estimation of the status of the ghrelin system. Interestingly, we found that plasma levels of ghrelin and DAG showed no gender differences in any condition, in line with previous studies in children [START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Razzaghy-Azar | An evaluation of acylated ghrelin and obestatin levels in childhood obesity and their association with insulin resistance, metabolic syndrome, and oxidative stress[END_REF] and adults [START_REF] Shiiya | Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion[END_REF][START_REF] Tschop | Circulating ghrelin levels are decreased in human obesity[END_REF]. To our knowledge, plasma ghrelin + DAG levels show sexual dimorphism only in adolescents, when levels are higher in females than in males [START_REF] Soriano-Guillen | Sex-related differences in the association of ghrelin levels with obesity in adolescents[END_REF].

We show here, for the first time that lower LEAP2 levels are detected in children with OW/OB. No gender differences were found in plasma LEAP2 levels. Such observation suggests that obesity affects not only the predominance of ghrelin forms but also other GHSR ligands. LEAP2 blocks ghrelin effects [START_REF] Kadmi | N-terminal liver-expressed antimicrobial Peptide 2 (LEAP2) region exhibits inverse agonist activity toward the ghrelin receptor[END_REF][START_REF] Wang | Identifying the binding mechanism of LEAP2 to receptor GHSR1a[END_REF]; thus, a reduction of LEAP2 levels in children with obesity would facilitate the orexigenic and diabetogenic effects of ghrelin. Interestingly, the levels of LEAP2 we detected [START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF]. In contrast, this study found that plasma LEAP2 levels were similar between subjects with NW and subjects with obesity that had a BMI lower than 40 kg/m 2 [START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF]. A significant elevation of plasma LEAP2 levels was only detected in patients with BMI higher than 40 kg/m 2 (32). Thus, plasma LEAP2 levels seem to be affected only under severe obesity conditions in adults. The reasons for the divergent results between adults and children are uncertain. DAG and ghrelin + DAG negatively correlated with BAZ only in children with OW/OB, in a genderindependent manner. LEAP2 levels also correlated with BAZ, and such correlation was significant only in male children and in children with NW. These observations suggest that the mechanisms that link the levels of DAG and LEAP2 to body weight are altered in children with OW/OB. Hierarchical clustering analysis also indicated that the interdependence of DAG and LEAP2 with other variables, including BAZ, was different in children with OW/OB. Notably, DAG levels and levels of ghrelin + DAG negatively correlated with age, in a gender-and BAZindependent manner. Similar results were previously reported by other studies in diverse populations, many of which include adolescents [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Whatmore | Ghrelin concentrations in healthy children and adolescents[END_REF][START_REF] Haqq | Serum ghrelin levels are inversely correlated with body mass index, age, and insulin concentrations in normal children and are markedly increased in Prader-Willi syndrome[END_REF][START_REF] Wilasco | Ghrelin, leptin and insulin in healthy children: relationship with anthropometry, gender, and age distribution[END_REF]. Indeed, levels of ghrelin + DAG negatively correlated with the pubertal stage [START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Whatmore | Ghrelin concentrations in healthy children and adolescents[END_REF]. Overall, clinical data indicate that the ghrelin system plays a more relevant role at younger ages, when its regulatory effects on GH secretion could be more important. In line with this notion, studies in mice show that endogenous ghrelin modulates GH secretory episodes primarily during the rapid phase of growth [START_REF] Hassouna | An early reduction in GH peak amplitude in preproghrelin-deficient male mice has a minor impact on linear growth[END_REF]. Still, it is worth mentioning that we found that levels of ghrelin, DAG or LEAP2 did not correlate with height (expressed as HAZ) in children, in agreement with other authors [START_REF] Whatmore | Ghrelin concentrations in healthy children and adolescents[END_REF], suggesting that the plasma levels of these peptides are not directly linked to growth. The extent to which alterations of the ghrelin system affect the growth of children with OW/OB remains uncertain.

DAG levels negatively correlated with insulin and HOMA index, in a BAZ-and gender-independent manner. Levels of ghrelin + DAG also negatively correlated with insulin and HOMA index, but these correlations were significant only in children with OW/OB, in a genderindependent manner. Levels of ghrelin did not correlate with insulin and HOMA index, similarly as previously reported [START_REF] Barazzoni | Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome[END_REF]. Current observations are in line with the idea of a crosstalk between DAG and glycaemic control, which appears to be independent of obesity, and also highlight the beneficial role of DAG in glycaemic control that, in turn, may be dependent or independent of the inhibitory role of DAG on ghrelin effects [START_REF] Benso | Metabolic effects of overnight continuous infusion of unacylated ghrelin in humans[END_REF][START_REF] Ozcan | Does des-acyl ghrelin improve glycemic control in obese diabetic subjects by decreasing acylated ghrelin levels?[END_REF][START_REF] Broglio | Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans[END_REF]. Regarding the lipid profile, we mainly found weak associations. LEAP2 levels negatively correlated with LDL-C, and such correlation only showed statistical significance in children with OW/ OB in a gender-independent manner. Thus, a potential role of LEAP2 in obesity-related dyslipidaemia should be further studied. DAG levels as well as ghrelin + DAG negatively correlated with TG, VLDL-C and LDL-C exclusively in females. DAG levels negatively correlated with LDL-C in females and positively correlated with LDL-C in males. Previous studies also reported similar weak correlations between circulating lipids and ghrelin, although no gender differences were reported [START_REF] Razzaghy-Azar | An evaluation of acylated ghrelin and obestatin levels in childhood obesity and their association with insulin resistance, metabolic syndrome, and oxidative stress[END_REF][START_REF] Park | Relationships between fasting plasma ghrelin levels and metabolic parameters in children and adolescents[END_REF][START_REF] Purnell | Ghrelin levels correlate with insulin levels, insulin resistance, and high-density lipoprotein cholesterol, but not with gender, menopausal status, or cortisol levels in humans[END_REF][START_REF] Zou | Factors associated with fasting plasma ghrelin levels in children and adolescents[END_REF][START_REF] Wadden | Serum acylated ghrelin concentrations in response to short-term overfeeding in normal weight, overweight, and obese men[END_REF]. The weakness of the detected associations limits the ability to provide any certain conclusion in this regard.

Figure 1

 1 Figure 1 Agglomerative hierarchical clustering and heatmap diagram of ghrelin, DAG, LEAP2, BAZ, insulin, TG, VLDL, LDL and HDL in NW children (panel A) and in children with OW/OB (panel B). The metabolic and anthropometric variables (top row) and participants (left) are clustered hierarchically. Each column represents a variable and each row represents a participant in the study. Intensity of yellow or red colour represents low or high values, respectively, of the normalized data. A full colour version of this figure is available at https ://do i.org /10.1 530/E

European Journal of Endocrinology 182:2 3 Clinical Study

  Revision of Research Protocols (CIRPI) of the Institute of Development and Paediatric Research (IDIP),

	A S Fittipaldi and others	Ghrelin LEAP2 in children with
		obesity
	Study participants This cross-sectional cohort included 82 children, 3-12 PROOF ONLY
	years old, without chronic medical illness, a personal
	history of diabetes, history of medical condition, or
	medication related to obesity or diabetes risk status.
	Children were born with gestational age >37 weeks and
	birth weight >2500 g. Birth weight and gestational age
	were obtained from hospital records. All participants
	underwent a baseline evaluation, including a complete
	medical and family history and physical examination,
	including Tanner staging by a paediatrician. The study
	included only children at pre-or early-pubertal stages
	(Tanner stage I, II or III) and females who had not reached
	the menarche. Weight and height measurements were
	performed by nutrition staff. Weight was measured
	with the participants barefoot and wearing minimal
	clothing, on a mechanical scale (Co. AR. Me. Series E9637,
	Argentina) with a resolution of 10 g and expressed as
	Z-score and gender based on World Health Organisation
	(WHO) references according to the age (WAZ) (36). Height
	was measured with a Harpenden-type wall-mounted
	stadiometer (Holtain Ltd., United Kingdom) with a
	resolution of 1 mm and expressed as Z-score and gender
	based on WHO references according to the age (HAZ)
	(36). BMI was calculated by dividing weight by height
	squared (kg/m 2 ) and expressed as Z-score and gender
	based on WHO references according to the age (BAZ)
		La Plata Children's Hospital, and conducted according to
		the Declaration of Helsinki guidelines and Argentinian
		legal provisions governing clinical research on humans.
		Written informed consent and assent were obtained from
		all participants, as appropriate. Participants were recruited
		from the obesity program of the Nutrition Service at
		Sor María Ludovica Children's Hospital and from Elina
		de la Serna Children's Hospital (La Plata, Buenos Aires,
		Argentina), from April to December 2018.
		https://eje.bioscientifica.com

Table 1

 1 Baseline characteristics of patients.Data are presented as mean ± s.d. for age, glucose, total cholesterol, HDL-C and LDL-C. Insulin and HOMA index are presented as geometric mean (95% CI). BAZ, VLDL-C and TG are presented as median (IQR). Bold values are statistically significant (P < 0.05).

		NW	OW/OB	P value
	Age (years)	7.56 ± 2.41	8.12 ± 2.54	0.269
	Birthweight (g)	3491.4 ± 352.1	3474.2 ± 524.2	0.860
	WAZ	-0.42 ± 0.74	2.61 ± 1.51	<0.0001
	HAZ	-0.60 ± 1.09	0.51 ± 1.10	<0.0001
	BAZ	0.33 (-0.35; 0.60)	2.85 (1.92; 3.51)	<0.0001
	Insulin (µIU/mL)	2.95 (2.38; 3.64)	7.60 (5.74; 10.07)	<0.0001
	Glucose (mmol/L)	4.45 ± 0.43	4.52 ± 0.49	0.508
	HOMA index	0.58 (0.46; 0.72)	1.52 (1.14; 2.03)	<0.0001
	Total cholesterol (mg/dL)	1.57 ± 0.22	1.56 ± 0.37	0.898
	HDL-C (mg/dL)	0.48 ± 0.09	0.44 ± 0.09	0.037
	LDL-C (mg/dL)	0.91 ± 0.24	0.93 ± 0.33	0.817
	VLDL-C (mg/dL)	0.15 (0.12; 0.19)	0.16 (0.12; 0.24)	0.121
	TG (mg/dL)	0.74 (0.58; 0.93)	0.80 (0.61; 1.17)	0.081

BAZ, BMI z-score; HAZ, height z-score; HDL-C, high-density lipoprotein cholesterol; HOMA, homeostatic model assessment; LDL-C, low-density lipoprotein cholesterol; TG, triglycerides; VLDL-C, very-low-density lipoprotein cholesterol; WAZ, weight z-score.

Table 2

 2 Plasma levels of ghrelin, DAG and LEAP2 and their indices in all the subjects and in male and female children.

		NW			OW/OB			ANOVA P value
		All	Male	Female	All	Male	Female	Gender	BAZ
	Ghrelin (fmol/mL)	7.55 (5.75;	7.17 (5.00;	8.10 (4.88;	6.97 (5.72;	7.58 (5.37;	6.49 (5.04;	0.8340	0.6070
		9.61)	9.74)	12.14)	8.35)	10.16)	8.12)
	DAG (fmol/mL)	42.10	42.74 (33.80;	41.22 (28.52;	23.95	22.80 (16.76;	24.93	0.8550	0.0001
		(34.88;	52.71)	56.25)	(18.97;	29.77)	(17.15;
		50.00)			29.50)		34.17)
	Ghrelin + DAG (fmol/	51.09	51.00 (41.59;	51.21 (38.18;	32.33	31.82 (25.59;	32.76	0.8850	0.0001
	mL)	(43.63;	61.38)	66.14)	(27.17;	38.73)	(24.62;
		59.13)			37.94)		42.06)
	LEAP2 (pmol/mL)	2.98 (2.62;	3.06 (2.57;	2.88 (2.31;	2.32 (2.01;	2.43 (2.10;	2.24 (1.75;	0.472	0.0135
		3.40)	3.64)	3.59)	2.68)	2.81)	2.86)
	Ghrelin/DAG ratio	0.17 (0.12;	0.16 (0.11;	0.18 (0.10;	0.30 (0.22;	0.33 (0.20;	0.29 (0.17;	0.929	0.0151
		0.23)	0.23)	0.33)	0.43)	0.55)	0.47)
	Ghrelin/LEAP2 ratio	2.21 (1.70;	2.10 (1.51;	2.37 (1.48;	2.68 (2.07;	2.76 (1.91;	2.62 (1.78;	0.8960	0.2980
	(×10 -3 )	2.87)	2.92)	3.80)	3.47)	3.97)	3.86)

Data are presented as mean (95% CI) for ghrelin, DAG and ghrelin + DAG. Ghrelin/DAG ratio, LEAP2 and ghrelin/LEAP2 ratio are presented as geometric mean (95% CI). As the interaction term gender*BAZ was not significant in any model, it was excluded from ANOVA. ANOVA P values in bold are statistically significant (P < 0.05). DAG, des-acyl ghrelin; LEAP2, liver antimicrobial peptide 2. https://eje.bioscientifica.com correlated with LDL-C levels only in children with OW/ OB.

Table 3

 3 Correlations between DAG and LEAP2 and calculated indices with age, BAZ, insulin, HOMA index and LDL-C in all the subjects and in children with NW or OW/OB.

		Group	Ghrelin	DAG	Ghrelin + DAG	LEAP2	Ghrelin/DAG	Ghrelin/LEAP2
	Age	All	-0.01	-0.58	-0.58	0.04	0.40	-0.04
		NW	0.06	-0.51	-0.50	-0.11	0.33	0.12
		OW/OB	-0.07	-0.66	-0.66	0.14	0.47	-0.16
	BAZ	All	-0.1	-0.48	-0.50	-0.35	0.27	0.05
		NW	-0.29	-0.14	-0.26	-0.40	-0.15	-0.10
		OW/OB	-0.14	-0.32	-0.33	-0.07	0.19	-0.13
	Insulin	All	0.08	-0.59	-0.52	0.03	0.48	0.07
		NW	0.00	-0.34	-0.24	0.10	0.19	-0.03
		OW/OB	0.16	-0.59	-0.50	0.25	0.56	0.01
	HOMA	All	0.1	-0.59	-0.51	0.04	0.49	0.07
	index
		NW	0.00	-0.35	-0.25	0.08	0.20	-0.05
		OW/OB	0.18	-0.60	-0.50	0.27	0.57	0.01
	LDL-C	All	-0.04	0.01	0.00	-0.26	-0.02	0.15
		NW	-0.10	0.18	0.13	-0.20	-0.20	0.16
		OW/OB	0.02	-0.08	-0.08	-0.31	0.07	0.19
	HDL-C	All	-0.02	0.15	0.13	-0.11	0.16	0.06
		NW	-0.09	-0.24	-0.30	-0.10	-0.11	-0.02
		OW/OB	-0.11	0.34	0.31	-0.21	0.27	0.14

Bold values are statistically significant (P < 0.05). BAZ, BMI z-score; DAG, des-acyl ghrelin; HDL-C, high-density lipoprotein cholesterol; HOMA, homeostatic model assessment; LDL-C, low-density lipoprotein cholesterol; LEAP2, liver antimicrobial peptide 2; NW, normoweight; OW/OB, overweight/obese. https://eje.bioscientifica.com

Table 4

 4 Correlations between DAG and LEAP2 and calculated indices with age, BAZ, insulin, HOMA index, VLDL-C, LDL-C and TG in all the subjects and in male and female children.

		Group	Ghrelin	DAG	Ghrelin + DAG	LEAP2	Ghrelin/DAG	Ghrelin/LEAP2
	Age	All	-0.01	-0.58	-0.58	0.04	0.40	-0.04
		Male	0.00	-0.56	-0.61	0.01	0.31	-0.03
		Female	-0.05	-0.60	-0.57	0.07	0.46	-0.07
	BAZ	All	-0.10	-0.48	-0.50	-0.35	0.27	0.05
		Male	-0.04	-0.56	-0.59	-0.45	0.31	0.15
		Female	-0.20	-0.38	-0.43	-0.25	0.23	-0.08
	Insulin	All	0.08	-0.59	-0.52	0.03	0.48	0.07
		Male	0.06	-0.57	-0.55	0.02	0.38	0.05
		Female	0.03	-0.60	-0.55	0.09	0.55	0.03
	HOMA index All	0.10	-0.59	-0.51	0.04	0.49	0.07
		Male	0.08	-0.59	-0.55	-0.02	0.40	0.06
		Female	0.07	-0.58	-0.51	0.09	0.56	0.06
	VLDL-C	All	-0.09	-0.21	-0.19	0.02	0.12	-0.08
		Male	-0.20	0.03	0.01	0.18	-0.18	-0.26
		Female	-0.03	-0.44	-0.41	-0.03	0.42	0.00
	LDL-C	All	-0.04	0.01	0.00	-0.26	-0.02	0.15
		Male	-0.23	0.34	0.23	-0.22	-0.41	-0.09
		Female	0.17	-0.32	-0.26	-0.28	0.39	0.30
	HDL-C	All	-0.02	0.15	0.13	-0.11	0.16	0.06
		Male	0.03	0.12	0.11	-0.13	0.07	0.10
		Female	-0.05	0.15	0.15	-0.10	0.22	-0.23
	TG	All	-0.09	-0.17	-0.15	-0.05	0.08	-0.04
		Male	-0.21	0.02	0.00	0.12	0.19	-0.25
		Female	0.03	-0.34	-0.33	0.13	0.33	0.07

Bold values are statistically significant (P < 0.05). BAZ, BMI z-score; DAG, des-acyl ghrelin; HDL-C, high-density lipoprotein cholesterol; HOMA, homeostatic model assessment; LDL-C, low-density lipoprotein cholesterol; LEAP2, liver antimicrobial peptide 2; TG, triglycerides; VLDL-C, very low-density lipoprotein cholesterol.

PROOF ONLY

https://eje.bioscientifica.com in children with NW are similar to the levels recently reported in adults with NW

Funding

This work was supported by grants from the Fondo para la Investigación Científica y Tecnológica (FONCyT, PICT2016-1084, PICTO2017-0086 and PICT2017-3196), from CONICET (PUE-2017), from Fundación Alberto Roemmers and from the Commission for Scientific Research of the Province of Buenos Aires (FCCIC16) to M P. A S F was supported by CONICET.

PROOF ONLY

Here, we compared ghrelin/LEAP2 and ghrelin/ DAG ratios to determine their degree of association with different variables. The ghrelin/DAG ratio has been widely linked to obesity and insulin resistance [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Bellone | Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels[END_REF][START_REF] Barazzoni | Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome[END_REF][START_REF] Inhoff | Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats[END_REF][START_REF] Vivenza | Ghrelin gene polymorphisms and ghrelin, insulin, IGF-I, leptin and anthropometric data in children and adolescents[END_REF]. In agreement with our results, several authors reported that this ratio is increased in patients with obesity, either adults [START_REF] Barazzoni | Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome[END_REF], children or adolescents [START_REF] Kuppens | Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome[END_REF][START_REF] Vivenza | Ghrelin gene polymorphisms and ghrelin, insulin, IGF-I, leptin and anthropometric data in children and adolescents[END_REF]. The increase of the ghrelin/DAG ratio occurs as a consequence of the reduction of DAG levels, an unbalance that would facilitate the orexigenic and diabetogenic effects of ghrelin in children with OW/OB, as previously discussed for LEAP2. As expected, the ghrelin/DAG ratio positively correlated with variables that negatively correlate with DAG. In particular, ghrelin/DAG ratio positively correlated with age (in a gender-and BAZ-independent manner), with BAZ (exclusively in male children, in a BAZ-independent manner) and with insulin levels and HOMA index (in the whole population, and in children with OW/OB, in a gender-independent manner). In adults, the observation that ghrelin/DAG ratio correlates with insulin and HOMA index only in patients with OW/OB has been proposed as an indication that the ghrelin/DAG ratio contributes to modulate insulin action in obese conditions [START_REF] Barazzoni | Relationships between desacylated and acylated ghrelin and insulin sensitivity in the metabolic syndrome[END_REF]. Since LEAP2 blocks constitutive and ghrelin-evoked GHSR signalling [START_REF] Kadmi | N-terminal liver-expressed antimicrobial Peptide 2 (LEAP2) region exhibits inverse agonist activity toward the ghrelin receptor[END_REF], we hypothesized that the ghrelin/LEAP2 ratio could be useful to estimate ghrelin tone in children with obesity. Notably, the above referred study assessing LEAP2 in adults found lower plasma ghrelin and higher plasma LEAP2 levels in patients with severe obesity and, as a consequence, the ghrelin/LEAP2 ratio was decreased in these patients [START_REF] Mani | LEAP2 changes with body mass and food intake in humans and mice[END_REF]. In contrast, we found that the ghrelin/ LEAP2 ratio remained unaffected in children with OW/ OB, in a gender-independent manner, and it showed no correlation with any of the metabolic or anthropometric variables studied. Thus, the ghrelin/LEAP2 ratio did not provide any additional information in children.

In summary, we used specific commercial enzyme immunoassays to assess ghrelin, DAG and LEAP2 in plasma samples, which were carefully collected and stored, from fasted children with NW or OW/OB. We found that ghrelin levels did not differ between both groups, while DAG and LEAP2 decreased in children with OW/OB. Thus, we conclude that ghrelin tone is increased in childhood obesity, due to a decrease of plasma levels of DAG and LEAP2. Also, we found that DAG is associated to insulin resistance, particularly in children with OW/ OB. Notably, no highly relevant gender differences were found.

Declaration of interest

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

Author contribution statement M F Andreoli and M Perello contributed equally to this work. P V, J A F, A F, M F A, M P designed the study. A S F, J H, D C, D L, P N D F, V G, P V, M V F, J A F, A F, M F A, M P collected, analysed and/or interpreted the data. M F A and M P wrote the manuscript. All authors approved the submitted and published versions. Q4 Please check that only approved species-specific gene and protein nomenclature is used in your paper (human genes as per HUGO database: in uppercase italics, human proteins in uppercase roman; mouse/rat as per the MGI database: genes with initial uppercase italics, mouse/rat proteins all uppercase roman). For more details, see https://eje.bioscientifica.com/page/author/author-guidelines#genes and correct if necessary. Q5 Please check and approve the identification of the section level headings. Q6 We have been unable to find the DOI for these references; please provide if possible.

PROOF ONLY