B Segrestin

Eric Blond

MD e Philippe Cristini

M Laville

MD-PhD Emmanuel Disse

Iceta Sylvain
email: sylvain.iceta@chu-lyon.fr.

Md-Msc

PhD Kevin Seyssel

MD c , Bérénice Segrestin MD-MSc Stéphanie Lambert- Porcheron

Emilie Blond Md-Phd

Martine Laville Md-Phd

Dr Sylvain Iceta

Ghrelin concentration as an indicator of eating-disorder risk in obese women

Keywords: Drive for thinness, Eating disorders, Ghrelin, Impulsive behaviour, Obesity, Vulnerability

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

Obesity remains a major concern in patients with diabetes, and the management of obese patients is also still difficult, especially when associated with eating disorders (EDs) or disordered eating (DE). EDs have primarily been considered a psychiatric condition, whereas obesity has been regarded predominantly as a medical concern. However, there is now a widespread movement to consider EDs, DE and obesity as part of the same continuum of weight-related disorders. The basic components necessary for an integrated theory of DE and obesity include sociological and psychological parameters leading to the perception that 'I am too fat' is a problem, maladaptive limitation of food intake (dietary restraint) to correct the problem, hunger (from eating less food) and the physiological reaction to hunger (hormonal control of food intake). To enhance preventative and interventional efforts, there is a strong need for early detection of psychological factors contributing to the development and maintenance of EDs and obesity.

Food intake is tightly controlled in humans by homoeostatic and non-homoeostatic mechanisms that maintain stable body weight and neutral energy balance [START_REF] Nakazato | A role for ghrelin in the central regulation of feeding[END_REF]. Ghrelin, a 28 amino-acid peptide synthesized by the stomach, is a leading player in this physiological regulation [START_REF] Kojima | Ghrelin is a growthhormone-releasing acylated peptide from stomach[END_REF]. The acylated form of ghrelin (AG) stimulates appetite and food intake [START_REF] Kojima | Ghrelin is a growthhormone-releasing acylated peptide from stomach[END_REF] by activating neuropeptide Y neurons in the arcuate nucleus of the hypothalamus and inhibiting the melanocortin pathway [START_REF] Cabral | Brain circuits mediating the orexigenic action of peripheral ghrelin: narrow gates for a vast kingdom[END_REF]. However, the action of ghrelin in the brain is not restricted to homoeostatic control of energy balance within the hypothalamus. Indeed, AG also induces reward-seeking behaviours for food, alcohol and other substance abuse [START_REF] Jerlhag | Systemic administration of ghrelin induces conditioned place preference and stimulates accumbal dopamine[END_REF][START_REF] Jerlhag | Ghrelin stimulates locomotor activity and accumbal dopamine-overflow via central cholinergic systems in mice: implications for its involvement in brain reward[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] Panagopoulos | The role of ghrelin in addiction: a review[END_REF]. Moreover, AG modulates the dopaminergic and serotoninergic pathways involved in the regulation of impulsivity [START_REF] Abizaid | Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite[END_REF][START_REF] Skibicka | Divergent circuitry underlying food reward and intake effects of ghrelin: dopaminergic VTA-accumbens projection mediates ghrelin's effect on food reward but not food intake[END_REF] and, when injected into the lateral hypothalamus of rodents, it increases both motor and choice impulsivity [START_REF] Anderberg | The Stomach-Derived Hormone Ghrelin Increases Impulsive Behavior[END_REF].

As ghrelin impacts food intake, food reward and impulsivity, its plasma concentrations may reflect certain behavioural traits. Overeating, increased food reward sensitivity and impulsivity are characteristics shared by many patients with obesity and/or EDs [START_REF] Waxman | A systematic review of impulsivity in eating disorders[END_REF] and, contrary to expectations, obese participants usually exhibit lower plasma ghrelin concentrations than their normal-weight counterparts [START_REF] Shiiya | Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion[END_REF][START_REF] Soriano-Guillén | Ghrelin levels in obesity and anorexia nervosa: effect of weight reduction or recuperation[END_REF][START_REF] Tschöp | Circulating ghrelin levels are decreased in human obesity[END_REF]. These low plasma ghrelin concentrations have been considered a physiological adaptation to the positive energy balance associated with obesity [START_REF] Tschöp | Circulating ghrelin levels are decreased in human obesity[END_REF], and the same adaptive phenomenon has been described in some EDs, including binge-eating [START_REF] Monteleone | Circulating ghrelin is decreased in non-obese and obese women with binge eating disorder as well as in obese non-binge eating women, but not in patients with bulimia nervosa[END_REF]. However, in clinical practice, a wide range of plasma ghrelin concentrations is observed in obese individuals, with some exhibiting unexpectedly high concentrations approaching those observed in normal-weight individuals.

Based on this clinical observation, our hypothesis is that patients with obesity and high concentrations of ghrelin might display greater feelings of hunger, a higher prevalence of DErelated psychopathology/behaviours and, especially, food impulsivity. Thus, the aim of the present study was to determine whether obese patients with unexpectedly high concentrations of ghrelin differ from those with low ghrelin concentrations in terms of hunger and behavioural traits leading to a DE risk. Initially, 108 women were included in the study. Five normal-weight participants were excluded for uncontrolled eating scores > 8 (n = 4) or contraindicated medication use (n = 1), while 13 obese participants were excluded for anxiety scores > 11 (n = 7), unstable body weight (n = 4), unknown diabetes (n = 1) or recent cessation of tobacco use (n = 1). A flow chart detailing participant recruitment is presented in Fig. 1.

Materials and Methods

Participants

Study design

Following their inclusion, participants had to arrive at the study nutrition research centre (Centre de Recherche en Nutrition Humaine Rhône-Alpes, CRNH-RA) at 0700 h after an overnight fast. Anthropometric parameters were assessed, and fasting blood samples drawn for acyl ghrelin (AG) and des-acyl ghrelin (DAG), leptin, glucose and insulin quantification.

Participants were then asked to complete three self-administered psychometric questionnaires: the validated French translation of the Eating Disorder Inventory (EDI)-2, the Three-Factor Eating Questionnaire (TFEQ) and the Hospital Anxiety and Depression Scale (HADS). Fasting hunger was evaluated at 0900 h using a 10-cm visual analogue scale (VAS), which rated hunger strength from 0 (not hungry) to 10 (extremely hungry). More precisely, participants had to answer the question 'How hungry do you feel at this moment?' by placing a mark along the VAS.

Self-administered questionnaires

The Eating Disorder Inventory (EDI)-2 is a 91-item multidimensional self-reported questionnaire that assesses attitudes, feelings and behaviours typically associated with EDs.

Although 11 subscales were used in this study, there was a particular interest in the five subscales that could be affected by appetite, feelings of satiety and food intakes: drive for thinness; bulimia; body dissatisfaction; interoceptive awareness; and impulse regulation.

The Three-Factor Eating Questionnaire (TFEQ) is a food-intake-behaviour questionnaire [START_REF] Stunkard | The three-factor eating questionnaire to measure dietary restraint, disinhibition and hunger[END_REF] comprising 51 items that measure three dimensions of human eating behaviour: cognitive restraint (21 items); uncontrolled eating (16 items); and emotional eating (14 items). Each item has a binary score (0 or 1) that is summed with the others, and each subscore corresponds to the three dimensions of eating behaviour: the higher the score, the more problematic the related behaviour. Uncontrolled eating scores > 8 were an exclusion criterion for the normal-weight participants in this study.

The Hospital Anxiety and Depression Scale (HADS) is a questionnaire [START_REF] Snaith | Anxiety and depression in general medical settings[END_REF] of 14 items commonly used to determine the probability of anxiety (seven items) and depression (seven items). Each item has a score between 0 and 3: the higher the score, the higher the probability of exhibiting anxiety or depression. The threshold of each subscore to detect the presence of anxiety or depressive symptoms is 8/21.

Blood samples

Venous blood was collected in EDTA tubes and centrifuged immediately for 10 min at 4° C at 4500 rpm. Plasma glucose was determined by the hexokinase method using an Architect ci8200 clinical chemistry analyzer (Abbott Laboratories, Abbott Park, IL, US). Insulin concentrations were determined by immunoradiometric assay using Bi-Ins-IRMA kits (IBA Molecular -Cis Biointernational, Gif-sur-Yvette, France). For AG determinations, 1 mM phydroxymercuribenzoic acid in HCl was added to blood samples to prevent protease degradation of AG (as recommended by Cayman Chemical, Ann Arbor, MI, USA). Plasma samples were then immediately stored at < -20° C until needed for analysis. Plasma AG and DAG concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using SPI-Bio kits (Bertin Pharma, Montigny-le-Bretonneux, France), and leptin was also measured by ELISA (Clinical Range, BioVendor, Brno, Czech Republic). Homoeostasis model assessment of insulin resistance (HOMA-IR) was used as a surrogate marker of insulin resistance and calculated as glucose (mmol.L -1 ) × insulin (mIU.L -1 )/22.5.

Stratification of obese participants according to fasting plasma AG concentrations

Plasma AG values were available for 84/90 participants, and the six participants lacking AG values were excluded from the analysis. The severely obese participants were stratified into two groups according to the median AG value obtained in this sample.

Ethics

All participants gave their written informed consent to participate. The study promoter was the Hospices Civils de Lyon (University Hospital), and the study was performed after a favourable opinion from the Committee for the protection of persons concerned (CPP) in accordance with French law (Data Protection and Civil Liberties) and the ethical standards laid down by the 1964 Declaration of Helsinki. The ClinicalTrials.gov identifier is NCT01948414.

Statistical analysis

Data were analyzed using SPSS version 19.0 software (IBM Corp., Armonk, NY, USA) and expressed as the mean ± SEM (standard error of mean). Normality of each variable was checked using the Shapiro-Wilk test. Differences between normal-weight (NW) and obese (OB) participants were assessed by a parametric (t) test or a non-parametric test (Mann-Whitney U test, for non-normal distributions). Anthropometric, biological and psychometric differences among the three groups (NW and two OB groups) were assessed using one-way analysis of variance (ANOVA) or by Kruskal-Wallis test. Multiple comparisons were performed using Tukey's post-hoc test, while effect sizes were assessed by Cohen's d. Linear correlations between AG and drive-for-thinness (DT) scores were assessed by the Pearson correlation coefficient. For all statistical analyses, the level of significance was the conventional two-sided 5% alpha error.

Results

Participants' characteristics

These are presented in Table I. The population included 29 NW and 55 severely OB (BMI ≥ 35 kg.m -2 ) women. The severely OB women were stratified into two groups according to the median AG value observed in this sample (19.0 pg.mL -1 ; 95% CI: 12.0-44.0; Fig. 1): 28 participants were allocated to the low ghrelin concentration (LGC) group (mean AG value: 7.51 ± 1.11 pg.mL -1 ); and 27 were allocated to the high ghrelin concentration (HGC) group (mean AG value: 65.77 ± 8.67 pg.mL -1 ).

The three groups did not differ in either age or height, although OB participants exhibited higher systolic and diastolic blood pressures than did NW participants. Stratification of the OB group according to fasting plasma AG concentration revealed no anthropometric differences between the LGC and HGC groups.

Biological profiles

These profiles are presented in Table I and, compared with those in the NW group, women in the OB groups had higher fasting glucose and insulin values, and higher levels of insulin resistance, whereas there were no significant differences in metabolic parameters between the HGC and LGC subgroups. Leptin was higher, whereas fasting AG, DAG and total ghrelin levels were lower in the OB vs NW groups, although leptin did not differ between the LGC and HGC groups. In the OB groups, mean fasting plasma AG was 36.11 ± 5.8 pg.mL -1 and the median was 19.0 pg.mL -1 . AG values in these OB groups were not normally distributed and, as shown in Fig. 2, a bimodal distribution of ghrelin in this sample corresponded to the HGC and LGC subgroups. Also in keeping with the study design, significant differences in plasma ghrelin concentrations were observed between LGC, HGC and NW groups (ANOVA-1; P < 0.001). The HGC subgroup had a fasting plasma AG concentration approaching that of the NW group, but nevertheless remained significantly lower than that of the NW group (Table I).

Hunger scores

Fasting hunger scores were similar between OB and NW groups, although a trend was observed towards higher hunger scores in the HGC vs LGC groups (4.51 ± 0.46 vs 3.38 ± 0.43, respectively; P = 0.08) with a medium effect size (d = 0.49).

Self-administered questionnaire scores

The EDI-2, TFEQ and HADS scores are presented in Table II. Psychological states assessed by HADS showed similar anxiety scores in both the NW and OB groups, but a significantly greater depression score in the OB groups (P = 0.008). However, no difference in anxiety and depression scores was found between the HGC and LGC subgroups.

As for the TFEQ, both OB groups scored significantly higher in uncontrolled and emotional eating (P < 0.001; Fig. 3A, Table II) than did the NW group, although there was no significant difference in cognitive restraint. Also, no significant difference was found in the three subscales of the TFEQ between the LGC and HGC subgroups (Fig. 3B), although a small effect size was observed for cognitive restraint (d = 0.19; Table II). Behavioural profiles frequently associated with EDs were assessed by EDI-2 (Table II), and almost all subscales, especially the five of particular interest (Fig, 3C), differed between the OB and NW groups, except for the 'maturity fears' subscale, albeit not significantly (P > 0.05; Table II). The LGC and HGC subroups differed significantly only for the DT subscale (Table II, Fig. 3D): OB participants in the HGC group had a higher DT score than the LGC group (9.30 ± 0.99 vs 6.46 ± 0.83, respectively; P < 0.05). A medium effect size was observed between groups for DT (d = 0.63), whereas there were small effect sizes for body dissatisfaction (d = 0.39) and interpersonal distrust (d = 0.36), although both were higher in the HGC group. In addition, a positive and significant linear correlation was observed between AG values and DT scores in the entire OB population (rho = 0.297, P = 0.03).

Discussion

The present study confirms our assumption, based on clinical experience, that fasting AG values are wide-ranging in OB participants, with some of them having unexpectedly high concentrations of ghrelin approaching those observed in their NW counterparts. This observation might be a reflection of the variability in central ghrelin resistance associated with obesity, a feature that has also been demonstrated in animal models of obesity [START_REF] Andrews | Central mechanisms involved in the orexigenic actions of ghrelin[END_REF][START_REF] Zigman | Obesity Impairs the Action of the Neuroendocrine Ghrelin System[END_REF].

However, high fasting concentrations of AG in OB participants were indicative of higher DT subscale scores in the EDI-2 and a trend towards higher hunger scores.

The concept of DT includes aspiring to a thin body ideal, thereby placing excessive importance on thinness while experiencing extreme fear of becoming fat. It is considered a key motivational variable underlying dieting/weight-loss efforts [START_REF] Rodin | Women and weight: a normative discontent[END_REF][START_REF] Polivy | Etiology of binge eating: Psychological mechanisms[END_REF]. While DT may reflect a healthy motivation to lose weight in obesity, other studies of OB binge-eaters, using the EDI-2, have found that DT is also a strong predictor of ED severity [START_REF] Hawkins | Development and construct validation of a self-report measure of binge eating tendencies[END_REF][START_REF] Lowe | Binge eating in obesity: Toward the specification of predictors[END_REF].

In the present study, all EDI-2 scores remained under the cut-off values usually considered indicative of the presence of an ED (DT > 14, body dissatisfaction > 16, bulimia > 8) [START_REF] Garner | Eating disorder inventory-2. psychological assessment resources Odessa[END_REF][START_REF] Penas-Lledo | Subtyping eating disordered patients along drive for thinness and depression[END_REF][START_REF] Rosen | Psychological adjustment of adolescents attempting to lose or gain weight[END_REF]. However, it has been shown that DT scores are more relevant to binge-eating disorder (BED) than to obesity [START_REF] Villarejo | Loss of Control over Eating: A Description of the Eating Disorder/Obesity Spectrum in Women[END_REF]. Yet, little data are available in the scientific literature on the links between DT and obesity other than BED. Nevertheless, it has been reported in previous research that a higher DT is associated with bigger body size [START_REF] Allaz | Body weight preoccupation in middle-age and ageing women: a general population survey[END_REF][START_REF] Pingitore | Gender differences in body satisfaction[END_REF] and with more intentional weight-loss behaviours [START_REF] Keski-Rahkonen | Intentional weight loss in young adults: sex-specific genetic and environmental effects[END_REF]. Obesity is associated with body dissatisfaction, leading some patients to have a high desire for thinness which can, in turn, trigger intentional weight-loss behaviours with repetitive food restrictions that can induce EDs such as BED [START_REF] Polivy | Dieting and binging. A causal analysis[END_REF]. Given this possibility, it is not surprising to find an association between DT and BED.

Indeed, in a 2016 study by Mustelin et al. [START_REF] Mustelin | The Eating Disorder Inventory in the screening for DSM-5 binge eating disorder[END_REF], it was proposed that the DT and other EDI-2 subscales could be useful in combination for the diagnosis of BED, in particular, the drive for thinness, bulimia and body dissatisfaction subscales. The fact that our study focused on patients without EDs and our final sample size might explain why those dimensions did not differ significantly between our HGC and LGC subgroups.

In the available literature thus far, only a few studies have focused on ghrelin concentrations and the DT subscale. In women who exercise and in women with anorexia, high DT scores have been associated with significantly higher ghrelin concentrations in comparison to normal DT groups, independently of BMI [START_REF] Polivy | Etiology of binge eating: Psychological mechanisms[END_REF][START_REF] Hawkins | Development and construct validation of a self-report measure of binge eating tendencies[END_REF]. Those authors considered that high ghrelin concentrations were adaptations for chronic energy deficiencies.

Although ghrelin has been a topic of DE research for > 10 years, its causal involvement has still not been resolved. It remains unclear whether ghrelin plays a functionally important role in the aetiology of DE and EDs. Common polymorphisms in human ghrelin and ghrelin receptor genes are unlikely to represent major contributors to the pathogenesis of obesity [START_REF] Del Giudice | Molecular screening of the ghrelin gene in Italian obese children: the Leu72Met variant is associated with an earlier onset of obesity[END_REF]. In one large case-control study, three common single nucleotide polymorphisms (SNPs) in the human ghrelin gene were studied in 692 Caucasian cases with EDs (366 with anorexia nervosa and 326 with bulimia nervosa) and found no specific associations [START_REF] Cellini | Case-control and combined family trios analysis of three polymorphisms in the ghrelin gene in European patients with anorexia and bulimia nervosa[END_REF]. Concerning binge-eating, a positive correlation between fasting concentrations of plasma ghrelin and frequency of binge/vomiting episodes in bulimic women was reported, but not confirmed in later studies [START_REF] Monteleone | No association of the Arg51Gln and Leu72Met polymorphisms of the ghrelin gene with anorexia nervosa or bulimia nervosa[END_REF][START_REF] Troisi | Plasma ghrelin in anorexia, bulimia, and binge-eating disorder: relations with eating patterns and circulating concentrations of cortisol and thyroid hormones[END_REF][START_REF] Otto | Letter to the Editor: Similar fasting ghrelin levels in binge eating/purging anorexia nervosa and restrictive anorexia nervosa[END_REF]. In addition, the majority of studies reported lower plasma ghrelin concentrations in OB participants with BED, which was contrary to the expected higher concentrations [START_REF] Geliebter | Plasma ghrelin concentrations are lower in bingeeating disorder[END_REF].

Ghrelin is also involved in the regulation of impulsivity [START_REF] Abizaid | Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite[END_REF][START_REF] Skibicka | Divergent circuitry underlying food reward and intake effects of ghrelin: dopaminergic VTA-accumbens projection mediates ghrelin's effect on food reward but not food intake[END_REF][START_REF] Anderberg | The Stomach-Derived Hormone Ghrelin Increases Impulsive Behavior[END_REF]. Injecting ghrelin into the lateral ventricle of rodents increased both motor and choice impulsivity [START_REF] Anderberg | The Stomach-Derived Hormone Ghrelin Increases Impulsive Behavior[END_REF]. However, in the present study, impulsivity in the HGC subgroup, as assessed by the impulse regulation subscale of EDI-2 and the uncontrolled and emotional eating subscales of TFEQ, did not differ from that in the LGC group. An obesity-induced central ghrelin resistance, a phenomenon that has yet to be demonstrated in humans, might partly explain the absence of a strong association between plasma ghrelin concentrations and eating behaviours, especially food impulsivity, in the present study [START_REF] Zigman | Obesity Impairs the Action of the Neuroendocrine Ghrelin System[END_REF].

One limitation of our study is that the findings refer to a single morning measurement of plasma AG. As circulating concentrations of this orexigenic peptide fluctuate throughout the day [START_REF] Cummings | A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans[END_REF], the pathophysiological relevance of just this one measurement could be questioned.

In addition, it is possible that the measurement of ghrelin in peripheral blood is not an accurate reflection of its intracerebral concentrations or activity. Moreover, the use of self-administered questionnaires may have introduced a disorder underestimation bias through either the participants' underreporting or lack of sensitivity. The use of neurocognitive tests may have been more efficient in detecting subthreshold impairments in eating or impulsivity, but such tests are difficult to access in routine care. Finally, the unknown menstrual status of our participants is a potential limitation when interpreting our data.

Conclusion

The present results support the hypothesis of a potentially positive relationship between fasting plasma AG concentrations and the risk of developing EDs, regardless of mood and anxiety, in OB women. While no inferences can be made as regards the direction of causality, it can be concluded that OB woman with high fasting plasma AG may be prone to display the problematic traits commonly associated with EDs. Thus, it is possible to postulate that fasting plasma ghrelin might be a biomarker of vulnerability for developing DE and, potentially, EDs in obesity. This vulnerability should be assessed and managed to prevent the weight cycling wherein the excessive importance of eating, body shape and weight leads to strict dieting that, in turn, results in binge-eating and weight regain.

Figure legends

Women aged between 20

 20 and 55 years and with either normal weight [body mass index (BMI) 20-25 kg.m -2 ] or severe obesity (BMI ≥ 35 kg.m -2 ) were recruited prospectively between July 2013 and June 2015 through printed advertisements on notice boards at various sites of Lyon 1 University, including the cafeteria of the University Hospital and Department of Endocrinology and Nutrition (Groupement Hospitalier Sud, Hospices Civils de Lyon, Lyon). Exclusion criteria included being male, a smoker, psychotropic medication use and substance abuse according to the Diagnostic and Statistical Manual of Mental Disorders, 4 th Edition (DSM-IV), anxiety and depression [Hospital Anxiety and Depression Scale (HADS) subscale anxiety and/or depression scores > 11), medical illness, previous bariatric surgery, diabetes and ongoing pregnancy. Patients with previous or current obvious EDs (defined by DSM-5) as assessed by TFEQ and/or medical interview were also excluded. To avoid metabolic and hormonal disturbances linked to physical activity and diet, participants had to report stable weight and no change in physical activity over the past 3 months.

Fig. 1 .

 1 Fig. 1. Flow chart of participant recruitment into the study.

Fig. 2 .

 2 Fig. 2. Fasting plasma ghrelin distribution in obese women stratified into two groups according to the median acyl ghrelin value (19.0 pg.mL -1 , indicated by the vertical line in the

Fig. 3 .

 3 Fig. 3. Kiviat diagrams of the results of self-administered questionnaires: (A, B) results of the

Fig. 1 .Figure 3 :

 13 Fig. 1.

Figure 2 :

 2 Figure 2: Fasting plasma ghrelin distribution among the obese group. Obese participants

Table I

 I Clinical and biological characteristics and fasting hunger scores in the study population < 0.05 vs NW group; NS: not significant; LGC/HGC: low/high ghrelin concentration; SBP/DBP: systolic/diastolic blood pressure; HOMA-IR: homoeostasis model assessment of insulin resistance TableIIEating Disorder Inventory (EDI)-2, Three-Factor Eating Questionnaire (TFEQ) and Hospital Anxiety and Depression Scale (HADS) scores on psychometric evaluation of the study population

	Normal-weight (NW) women (n = 29) Mean ± SEM 37 ± 2.0 163.9 ± 1.0 57.8 ± 1.1 21.5 ± 0.4 78 ± 1.2 94 ± 0.8 0.83 ± 0.01 110 ± 1.7 71 ± 1.2 65 ± 1.9 4.8 ± 0.1 4.5 ± 0.4 0.95 ± 0.1 12.5 ± 1.3 107 ± 12 365 ± 32 471 ± 40 0.23 ± 1.7 3.86 ± 0.45 (NW) women Obese (OB) Obese (OB) women (n = 55) Mean ± SEM 38 ± 1.5 163.8 ± 0.8 111.5 ± 2.5 41.5 ± 0.8 119 ± 2.1 129 ± 1.9 0.91 ± 0.01 124 ± 1.5 75 ± 1.2 70 ± 1.6 5.1 ± 0.1 9.7 ± 0.7 2.21 ± 0.2 57.7 ± 2.9 36 ± 6 145 ± 15 181 ± 20 0.18 ± 1.8 3.93 ± 0.32 women (n = 29) (n = 55) OB vs CT Mean ± SEM Mean ± SEM P 2.14 ± 0.46 7.85 ± 0.67 < 0.001 0.10 ± 0.08 2.07 ± 0.40 < 0.001 4.21 ± 0.83 21.67 ± 0.86 < 0.001 1.83 ± 0.69 3.13 ± 0.52 0.011 1.31 ± 0.38 5.29 ± 0.57 < 0.001 1.76 ± 0.42 3.51 ± 0.46 0.019 0.76 ± 0.24 3.49 ± 0.58 0.001 4.93 ± 0.38 5.20 ± 0.31 0.59 2.83 ± 0.39 5.22 ± 0.45 < 0.001 1.24 ± 0.54 2.40 ± 0.49 0.031 1.79 ± 0.53 3.49 ± 0.44 0.002 6.45 ± 0.75 8.02 ± 0.43 0.055 3.41 ± 0.39 8.84 ± 0.44 < 0.001 2.07 ± 0.32 5.31 ± 0.47 < 0.001 5.72 ± 0.38 6.65 ± 0.37 0.11 2.52 ± 0.30 4.07 ± 0.38 0.008 * P Normal-weight Clinical characteristics Age (years) Height (cm) Weight (kg) Body mass index (kg.m -2 ) Waist circumference (cm) Hip circumference (cm) Waist-to-hip ratio SBP (mmHg) DBP (mmHg) Heart rate (bpm) Biological characteristics Glucose (mmol.L -1 ) Insulin (mIU.L -1 ) HOMA-IR Leptin (ng.mL -1 ) Acyl ghrelin (pg.mL -1 ) Des-acyl ghrelin (pg.mL -1 ) Total ghrelin (pg.mL -1 ) Acyl ghrelin/total ghrelin Visual analogue scale Hunger scale EDI-2 Drive for thinness Bulimia Body dissatisfaction Ineffectiveness Perfectionism Interpersonal distrust Interoceptive awareness Maturity fears Asceticism Impulse regulation Social insecurity TFEQ Cognitive restraint Uncontrolled eating Emotional eating HADS Anxiety score Depression score * P < 0.05 vs NW group; LGC/HGC: low/high ghrelin concentration	OB women grouped by fasting plasma acyl ghrelin concentration (n = 28) (n = 27) Mean ± SEM Mean ± SEM 37 ± 2.2 38 ± 2.1 162.9 ± 1.2 164.7 ± 1.1 113.4 ± 4.0* 109.5 ± 3.1* 42.6 ± 1.3* 40.3 ± 0.9* 122 ± 2.5* 116 ± 3.5* 129 ± 2.8* 128 ± 2.5* 0.94 ± 0.02* 0.89 ± 0.02* 126 ± 2.2* 123 ± 2.1* 76 ± 1.9 74 ± 1.6 74 ± 2.0* 66 ± 2.4 5.1 ± 0.1* 5.1 ± 0.1* 10.8 ± 1.0* 8.6 ± 0.8* 2.45 ± 0.25* 1.96 ± 0.18* 61.2 ± 4.3* 54 ± 3.8* 8 ± 1* 66 ± 9* 101 ± 8* 190 ± 27* 109 ± 8* 256 ± 33* 0.083 ± 1.5* 0.27 ± 2.0 3.38 ± 0.43 4.51 ± 0.46 OB women grouped by fasting plasma OB vs NW P NS NS < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.042 0.046 0.002 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.049 NS acyl ghrelin concentration LGC (n = 28) HGC (n = 27) LGC vs HGC LGC vs HGC LGC vs HGC P NS NS NS NS NS NS NS NS NS NS NS NS NS NS < 0.05 < 0.05 < 0.05 < 0.05 NS Mean ± SEM Mean ± SEM Cohen's d P 6.46 ± 0.83* 9.30 ± 0.99* 0.63 0.032 1.93 ± 0.61* 2.22 ± 0.53* 0.09 0.72 20.32 ± 1.32* 23.07 ± 1.06* 0.39 0.11 3.39 ± 0.78* 2.85 ± 0.69* 0.13 0.61 5.71 ± 0.91* 4.85 ± 0.70* 0.18 0.45 3.00 ± 0.54 4.04 ± 0.75* 0.36 0.26 3.32 ± 0.88* 3.67 ± 0.76* 0.07 0.77 5.25 ± 0.45 5.15 ± 0.44 0.04 0.87 5.46 ± 0.64* 4.96 ± 0.64* 0.15 0.58 2.43 ± 0.61 2.37 ± 0.79 0.02 0.95 3.64 ± 0.60* 3.33 ± 0.65* 0.10 0.72 7.68 ± 0.67 8.37 ± 0.55 0.19 0.43 8.89 ± 0.65* 8.78 ± 0.62* 0.03 0.90 5.25 ± 0.68* 5.37 ± 0.66* 0.03 0.89 6.75 ± 0.47 6.56 ± 0.57 0.08 0.79 4.61 ± 0.55* 3.52 ± 0.50 0.37 0.15

Acknowledgments

We thank the staff of the Centre de Recherche en Nutrition Humaine Rhône-Alpes for their helpful assistance. We also thank Philip Robinson for his writing assistance, language editing and proofreading.

Funding: This work was supported by the Young Investigator Award of the Hospices Civils de Lyon, France.

Conflict of Interest (COI) Statement: None to declare

Authors' contributions: ED, SLP and PC designed the research; BJ, BS, EB and SLP conducted the research; BJ, ED and KS analyzed the data or performed the statistical analyses; SI, BJ and ED wrote the paper; BS, SLP and ML performed a critical revision of the manuscript for important intellectual content. ED is primarily responsible for its final content; all authors read and approved the final version of the manuscript.


Amira S Khan

Babar Murtaza

Aziz Hichami

Naim Akhtar

Akhtar Khan

Amira S Khan

Naim A Khan
email: naim.khan@u-bourgogne.fr

Prof. N A Khan

A cross-talk between fat and bitter taste modalities

Keywords: Bitter taste, fat taste, cross-talk, genetic polymorphism

Introduction

Humans are omnivorous food-seeking species that have developed through evolution a sense of taste to survive in a complex environment. Taste is a sensory modality, involving the oral perception of food-derived chemicals that stimulate receptor cells within taste buds [START_REF] Shi | Adaptive diversification of bitter taste receptor genes in Mammalian evolution[END_REF].Taste not only helps decide whether a food is noxious or palatable but also prepares the gastrointestinal tract for post-ingestive metabolic events. The 5 basic taste modalities have been demonstrated. Simple carbohydrates are perceived as sweet, sodium salts and salts of few cations are perceived as salty, acids are perceived as sour while amino acids like glutamate and aspartate elicit a savoury taste [START_REF] Roper | Taste buds: cells, signals and synapses[END_REF]. Moreover, during the recent couple of years, there has been an increasing agreement that fatty acids might trigger a lipid taste [START_REF] Ozdener | CD36-and GPR120-mediated Ca 2+ signaling in human taste bud cells mediates differential responses to fatty acids and is altered in obese mice[END_REF].

Recent evidence indicates that CD36 and GPR120 might play a non-overlapping role in the detection of a fat taste [START_REF] Besnard | Taste of fat: a sixth taste modality?[END_REF].

The taste receptor (TR) cells are regrouped in taste buds which are the units of the papillae, distributed on the tongue. There are three kinds of papillae: fungiforme (localized in the frontal part of the tongue), foliate (localized in the lateral region of the tongue) and circumvallate (present on the anterior part of the tongue). There are three types of taste cells wherein type I cells are considered as supporting cells, type II cells respond to bitter, sweet and umami, and type III cells are involved in making synaptic junctions with afferent nerve fibres. In this review, we will not go into the details of perception of different taste modalities, as several recent review articles can be consulted on this subject [START_REF] Roper | Taste buds: cells, signals and synapses[END_REF][START_REF] Mennella | The development of sweet taste: From biology to hedonics[END_REF][START_REF] Murtaza | Alteration in taste perception in cancer: causes and strategies of treatment[END_REF][START_REF] Kinnamon | Taste transduction: a diversity of mechanisms[END_REF]. Briefly, the heterodimeric T1R2/T1R3 detects the sweet taste whereas, the T1R1/T1R3 heterodimer is sensitive to umami taste [START_REF] Roper | Taste buds: cells, signals and synapses[END_REF]. Salt activates sodium channels, while the acidic compounds induce depolarization by blocking potassium channels [START_REF] Kinnamon | Taste transduction: a diversity of mechanisms[END_REF]. TAS2R receptors have been shown to be involved in the perception of bitter taste. Bitter molecules belong to the largest family of tastants and humans possess more than 25 functional bitter taste receptor genes to interact with such large group of taste substances [START_REF] Shi | Adaptive diversification of bitter taste receptor genes in Mammalian evolution[END_REF]. Therefore, most of the noxious and poisonous substances are experienced as bitter.

The majority of laboratory studies on taste perception are based on the use of candidate molecules. However, in the real world, food, most often, comprises of a mixture of agents that can trigger different taste sensations at the same time. The sense of taste can interact with other senses, and different taste modalities may interact with each other [START_REF] Landis | Chemosensory interaction: acquired olfactory impairment is associated with decreased taste function[END_REF][START_REF] Noble | Taste-aroma interactions[END_REF]. For instance, salt taste has been shown to inhibit bitterness in food and enhances flavour [START_REF] Keast | A psychophysical investigation of binary bittercompound interactions[END_REF][START_REF] Remis | Taste responses to fructose and tannic acid among gorillas (Gorilla gorilla gorilla)[END_REF].

Similarly, salty and sour tastes have been shown to interact each other with the enhancement of their effect at low concentrations, while causing an inhibition or no effect at higher concentrations [START_REF] Keast | An overview of binary taste-taste interactions[END_REF]. It is interesting to mention that some amino acids like L-arginie or Llysine may modulate the oro-sensory perception of 5 taste modalities. The exogenous L-Arg was found to increase the perception of sucrose, umami, sodium chloride, salt and bitterness of caffeine only in tasters, and to decrease citric acid sourness [START_REF] Melis | Taste Perception of Sweet, Sour, Salty, Bitter, and Umami and Changes Due to L-Arginine Supplementation, as a Function of Genetic Ability to Taste 6-n-Propylthiouracil[END_REF]. Indeed, L-arginine has been considered as "carrier" as its addition to bitter molecules increases the bitterness as this agent makes hydrogen-bonded adducts that increase the solubility of PROP and its interaction with the receptor [START_REF] Melis | Dose-Dependent Effects of L-Arginine on PROP Bitterness Intensity and Latency and Characteristics of the Chemical Interaction between PROP and L-Arginine[END_REF]. A detailed understanding of various taste interactions is out of the scope of the current appraisal and can be found elsewhere [START_REF] Keast | An overview of binary taste-taste interactions[END_REF]. We will focus on the crosstalk between bitter and fat taste for the remaining part of this review.

Interaction between fat and bitter taste

Evidence from behavioural studies

To assess the bitter taste perception, the investigators have employed different agonists/agents like PROP (6-n-propylthiouracial), PTC (phenylthiocarbamide), quinine, caffeine etc. that elicit an unpleasant taste sensation [START_REF] Mueller | The receptors and coding logic for bitter taste[END_REF]. On the basis of sensitivity to one of the bitter compounds, the researchers have classified the subjects as taster, non-taster or super taster. As regards fat taste perception, the fatty acids like oleic acid or linoleic acid or sometimes oils like canola oil or fat emulsions have been used in rodents and/or humans [START_REF] Metcalf | Taste intensities of oil-in-water emulsions with varying fat content[END_REF][START_REF] Gaillard | The gustatory pathway is involved in CD36-mediated orosensory perception of long-chain fatty acids in the mouse[END_REF]. In this review, we will employ a general term "fat taste perception" that would mean the use of one of hence-mentioned lipids/fatty acids in the studies.

There are several studies that have reported that fat and bitter tastes might interact with each other. Duffy and Bartoshuk [START_REF] Duffy | Food acceptance and genetic variation in taste[END_REF] proposed that PROP supertasters may avoid high-fat food due to their intense sensations. Interestingly, PROP non-tasters were found to have high adiposity [START_REF] Tepper | Variation in the bitter-taste receptor gene TAS2R38, and adiposity in a genetically isolated population in Southern Italy[END_REF], and PROP phenotype can be used to discriminate different modalities of food preference including alcohol intake [START_REF] Duffy | Bitter receptor gene (TAS2R38), 6-n-propylthiouracil (PROP) bitterness and alcohol intake[END_REF]. Tepper and Nurse [START_REF] Tepper | PROP taster status is related to fat perception and preference[END_REF] reported that bitter non-taster participants were unable to accurately distinguish fat content in high and low-fat Italian salad dressings. Nasser et al. [START_REF] Nasser | PROP taster status and oral fatty acid perception[END_REF] have shown that the majority of PROP non-tasters were also less sensitive in discriminating fatty acids in a high-fat food. Hayes and Duffy [START_REF] Hayes | Revisiting sugar-fat mixtures: sweetness and creaminess vary with phenotypic markers of oral sensation[END_REF] demonstrated that PROP taste phenotype affects the nature of enhancement or suppression of creaminess in liquid fat/sugar mixtures.

On the contrary, eighty percent of the PROP tasters in this study correctly discriminated the sample containing the added free fatty acid. These observations suggest that bitter taste modality might interfere with lipid taste perception, though Drewnowski et al [START_REF] Drewnowski | Genetic sensitivity to 6-n-propylthiouracil has no influence on dietary patterns, body mass indexes, or plasma lipid profiles of women[END_REF] proposed that PROP responsiveness was not associated with the presence of low dietary fat in tasting sessions. However, there are reports demonstrating that the presence of fatty acids in tastingsessions influences oro-sensory perception of bitter compounds. Mattes et al. [START_REF] Mattes | Effects of linoleic acid on sweet, sour, salty, and bitter taste thresholds and intensity ratings of adults[END_REF] reported that the perceived intensity of high concentration of caffeine was rated weak in the presence of linoleic acid. Koriyama et al. [START_REF] Koriyama | Fatty acid compositions of oil species affect the 5 basic taste perceptions[END_REF] reported that tauna oil suppressed bitter taste perception, though sweetness or saltiness remained unaffected. Pittman et al. [START_REF] Pittman | Linoleic and oleic acids alter the licking responses to sweet, salt, sour, and bitter tastants in rats[END_REF] reported that the addition of linoleic and oleic acid to bitter taste solutions produced decreases in licking responses in rats when compared with the licking responses to the solutions in the absence of free fatty acids. Altogether, these studies indicate that bitter taste and fat taste might interfere with each other. A brief summary of the evident findings of the various studies cited above has been given in Table 1A.

Evidence from genetic polymorphism

In humans, bitter taste receptors are encoded by at least 25 TAS2R-genes [START_REF] Shi | Adaptive diversification of bitter taste receptor genes in Mammalian evolution[END_REF]. Some polymorphisms of these genes give rise to individual differences in bitter taste sensitivity.

Genetic association of bitter taste came into notice, for the first time, in 1930 when Arthur

Fox reported that PTC for some individuals was virtually tasteless [START_REF] Barbarossa | Variant in a common odorant-binding protein gene is associated with bitter sensitivity in people[END_REF][START_REF] Fox | The Relationship between Chemical Constitution and Taste[END_REF]. Afterwards, similar observations were also reported for PROP. The ability to taste bitter molecules containing a -N-C=S group "such as PTC and PROP" is inheritable. It is believed that PROP and PTC taste sensitivity follows a recessive Mendelian pattern of inheritance [START_REF] Yackinous | Relation between PROP taster status and fat perception, touch, and olfaction[END_REF]. In 2003, Kim et al. identified, for the first time, a small region on chromosome 7q which contained a single gene that encoded a member of the TAS2R bitter taste receptor family [START_REF] Kim | Positional cloning of the human quantitative trait locus underlying taste sensitivity to phenylthiocarbamide[END_REF]. The three single nucleotide polymorphisms (SNPs) give rise to 6 haplotypes (PAV, AVI, AAV, PAI and PVI) that are responsible for PTC and PROP sensitivity in the majority of population.

CD36 and GPR120 are two receptors that are implicated in the detection of fat taste in humans and rodents [START_REF] Besnard | Taste of fat: a sixth taste modality?[END_REF]. The CD36 gene spans 77 Kb (7q11.2-7q21.11) and is comprised of 9 alternatively spliced exons that are differentially regulated by several upstream promoters [START_REF] Cao | Structure and localization of the human gene encoding SR-BI/CLA-1. Evidence for transcriptional control by steroidogenic factor 1[END_REF]. The CD36 SNP is associated with Alzheimer's disease [START_REF] Šerý | CD36 gene polymorphism is associated with Alzheimer's disease[END_REF]. The team of Nada Abumrad reported, for the first time, the possible association of AA-genotype of CD36 rs1761667 with high detection thresholds for fatty acids in Afro-Americans subjects [START_REF] Pepino | The fatty acid translocase gene CD36 and lingual lipase influence oral sensitivity to fat in obese subjects[END_REF].

The same team further proposed that CD36 polymorphism is closely related to the disorders linked with excess of body lipids [START_REF] Love-Gregory | CD36 genetics and the metabolic complications of obesity[END_REF]. These studies have been confirmed in Algerian and Tunisian subjects by our team [START_REF] Mrizak | The A allele of cluster of differentiation 36 (CD36) SNP 1761667 associates with decreased lipid taste perception in obese Tunisian women[END_REF] wherein we have demonstrated that rs1761667-AA genotype is associated with obesity. It is noteworthy that rs1761667-AA genotype was also associated to de novo lipogenesis markers in obese subjects [START_REF] Melis | Polymorphism rs1761667 in the CD36 Gene Is Associated to Changes in Fatty Acid Metabolism and Circulating Endocannabinoid Levels Distinctively in Normal Weight and Obese Subjects[END_REF]. Interestingly, Plesnik et al. [START_REF] Plesník | The rs1527483, but not rs3212018, CD36 polymorphism associates with linoleic acid detection and obesity in Czech young adults[END_REF] reported that another variant of CD36, i.e., rs1527483 SNP, was associated with high body weight in young Czech participants.

The association between orosensory perception of oleic acid, rs1761667 SNP in the CD36 gene, and PROP tasting was, first time reported, by Melis et al. [START_REF] Melis | Associations between Orosensory Perception of Oleic Acid, the Common Single Nucleotide Polymorphisms (rs1761667 and rs1527483) in the CD36 Gene, and 6-n-Propylthiouracil (PROP) Tasting[END_REF] who showed that bitter non-taster subjects were also hyposensitive to fat taste, and CD36 SNP appeared to play a decisive role. The individuals who show reduced oral fat perception may consume high fat foods and, consequently, are more prone to become obese [START_REF] Keller | Genetic influences on oral fat perception and preference[END_REF]. The degree of decreased fat taste perception in PROP non-tasters had also been demonstrated in previous studies [START_REF] Hayes | Revisiting sugar-fat mixtures: sweetness and creaminess vary with phenotypic markers of oral sensation[END_REF][START_REF] Ebba | The examination of fatty acid taste with edible strips[END_REF][START_REF] Tepper | Genetic Sensitivity to the Bitter Taste of 6-n-Propylthiouracil (PROP) and Its Association with Physiological Mechanisms Controlling Body Mass Index (BMI)[END_REF][START_REF] Prescott | Prutkin, 6-n-Propylthiouracil tasting and the perception of nontaste oral sensations[END_REF][START_REF] Tepper | Fat perception is related to PROP taster status[END_REF], but a correlation with CD36 SNP was demonstrated for the first time by Melis et al. [START_REF] Melis | Associations between Orosensory Perception of Oleic Acid, the Common Single Nucleotide Polymorphisms (rs1761667 and rs1527483) in the CD36 Gene, and 6-n-Propylthiouracil (PROP) Tasting[END_REF].

The participants in this study were classified for their PROP taster status based on their ability to taste PROP, and genotyped for TAS2R38 locus, based on molecular analysis. TAS2R38 subjects (AVI/AVI) homozygous for the G-allele of the rs1761667 for CD36 had 11-fold lower detection threshold for oleic acid than subjects with the same TAS2R38 genotype (AVI/AVI), but with a homozygous AA genotype of the rs1761667 polymorphism.

PROP non-tasters exhibited a higher threshold for oleic acid than super-tasters. Hence, AA genotype of rs1761667 appeared to be associated with increased BMI (body mass index) and decreased sensitivity to fat and bitter taste perception [START_REF] Melis | Associations between Orosensory Perception of Oleic Acid, the Common Single Nucleotide Polymorphisms (rs1761667 and rs1527483) in the CD36 Gene, and 6-n-Propylthiouracil (PROP) Tasting[END_REF].

Our team has recently further investigated the association of PROP detection thresholds with CD36 and TAS2R38 genotypes in obesity in a Tunisian population [START_REF] Karmous | Orosensory detection of bitter in fat-taster healthy and obese participants: Genetic polymorphism of CD36 and TAS2R38[END_REF]. We observed that all the genotypes of rs1761667 (CD36) and rs1726866 (TAS2R38) and rs10246939 (TAS2R38), except AV genotype of rs1726866, were associated with higher PROP detection thresholds and higher BMI in obese subjects than normal weight participants. The participants with AA genotype of rs1726866 had significantly higher BMI than the subjects with AV and VV genotypes. High PROP thresholds in VV genotype of rs1726866 may also participate in obesity as V-allele of this genotype has been shown to be associated with high plasma leptin and increased disinhibition [START_REF] Dotson | Variation in the gene TAS2R38 is associated with the eating behavior disinhibition in Old Order Amish women[END_REF]. Interestingly, the obese participants with VV genotype of rs10246939 exhibited higher thresholds for LA (linoleic acid) than normal weight controls, and these VV genotype subjects had significantly higher BMI than the participants with IV and II genotypes. Also, the normal weight controls with VV genotype of rs10246939 showed significantly lower detection threshold than the control with II and IV genotypes. Hence, TAS2R38 variants rs1726866 and rs10246939 are associated with obesity. These SNP studies clearly suggest the relationship between fat and bitter taste perception in obesity.

Possible cross-talk between fat and bitter taste

Whether TAS2R38 plays a role as sensor for textural clues or it also provides chemosensory signals and is involved in cross-talk between fat and bitter taste is yet to be determined.

However, there are several reports that have shed light on the association between fat and bitter taste perception. Keller [START_REF] Keller | Genetic influences on oral fat perception and preference[END_REF] suggested that TAS2R38 might be involved in the textural perception of dietary fat through its association with the PROP taster phenotype, whereas CD36 protein would influence chemosensory perception of fat. However, the density of taste papillae is also variable among individuals and might play a crucial role in taste sensitivity [START_REF] Miller | Variation in human fungiform taste bud densities among regions and subjects[END_REF][START_REF] Zhang | The relationship between fungiform papillae density and detection threshold for sucrose in the young males[END_REF]. Some studies have suggested that bitter and fat taste sensitivity might be related to the density of taste papillae and the number of taste bud cells [START_REF] Feeney | Genetic variation in taste perception: does it have a role in healthy eating?[END_REF][START_REF] Scale | PROP (6-n-propylthiouracil) genetics and trigeminal innervation of fungiform papillae[END_REF]. Hence, the subjects containing high density of taste papillae might be more sensitive to oro-sensory detection of bitter and fatty acids. However, the relationship between the numbers of papillae and the intensity of taste perception is, sometimes, debated. For example, Hayes et al. [START_REF] Hayes | Supertasting and PROP bitterness depends on more than the TAS2R38 gene[END_REF] very clearly demonstrated that an alteration between decreased bitter taste perceptions was not proportional to the density of fungiform papillae. The concept of "bitter-like fat taste" seems to be very complicated and might involve other components. Barbarossa et al. [START_REF] Barbarossa | Variant in a common odorant-binding protein gene is associated with bitter sensitivity in people[END_REF] have recently reported that the presence of very dilute concentrations of oleic acid in milk shakes was associated with perceived bitterness in nearly half of the subjects and this bitterness was blunted remarkably when subjects were asked to wear nose-clips. Hence, olfactory stimulation appeared to play a critical role in the perception of "bitter taste" of fatty acids. These investigators proposed that the variability in the perceived perception of bitterness among individuals was associated with the genetic variability in the expression of an olfactory binding protein (OBPIIa) expressed in the olfactory cleft with high affinity for long chain fatty acids. The subjects homozygous for the A-allele for a common single nucleotide polymorphism (rs2590498) in the OBPIIa gene were able to perceive bitterness of oleic acid in the absence of nose clips. Subjects homozygous for G-allele for rs2590498 or heterozygous were unable to perceive bitterness of fatty acids, even when nose clips were omitted. Moreover, subjects homozygous for A-allele were also more sensitive to PROP than homozygous G-allele or heterozygous. These findings suggest the novel implication of olfaction in the cross talk-talk between bitter and fat tastes. Furthermore, a recent study by Lee et al. [53] has demonstrated that CD36 is expressed by olfactory receptor cells in mouse and plays a role in olfaction. It will be very interesting to investigate whether CD36 expressed on olfactory cells is also involved in the perception of a lipid taste through olfactory cues. Moreover, it will also be tempting to find whether a cross-talk exists between OBPIIa and CD36 expressed on olfactory cells. However, regardless of the mechanisms involved, the existence of an association between fat and bitter taste perception and obesity requires further alterations.

As for as the cellular and molecular interactions are concerned, we would like to state as follows. Both, fat (CD36 and GPR120) and bitter-taste receptors (TAS2Rs), are expressed on type-II cells which are gustducin-positive cells, suggesting that these cells might use gustducin for downstream signaling receptors [START_REF] Roper | Taste buds: cells, signals and synapses[END_REF][START_REF] Adler | A novel family of mammalian taste receptors[END_REF]. Binding of fat and bitter molecules to their respective receptors results in the activation of PLCβ which hydrolyses phosphatidylinositol 4, 5-bisphosphate (PIP 2) to inositol trisphosphate (IP 3 ). Subsequently, IP 3 binds to its receptor (IP 3 R) located on the endoplasmic reticulum (ER) and triggers the release of Ca 2+ (Figure 1). Hence, we can state that the common mechanism is "Ca 2+signaling" between the bitter and fat taste perception, though the later phenomenon is wellcharacterized at the molecular level, involving STIM1 and Orai1/3 Ca 2+ channels, for fat taste [START_REF] Dramane | STIM1 regulates calcium signaling in taste bud cells and preference for fat in mice[END_REF]. "The cross-talk" between the two taste modalities might also take place, downstream to Ca 2+ signaling via TRPM5 channels. Indeed, TRPM5 channels have been shown to be activated both in bitter and fat taste perception [START_REF] Liman | TRPM5 and taste transduction[END_REF]. Opening of TRPM5 channels results in the entry of Na + ions and depolarization of cell. In either case, the resulting cell depolarization succeeding an increase in intracellular concentrations of cations would result in the release of neurotransmitters, notably ATP and serotonin at afferent nerve endings which then would carry the taste message to sensory areas of the brain, involved in taste perception (Figure 1). Some bitter tastants like quinine and divalent salts might also block K + -channels and result in depolarization of the gustatory cell. Although, both fat and bitter taste modalities seem to be involved in cross-talk with each other, it is not known exactly how fat-taste might inhibit the bitterness perception which has been reported in various studies. Nonetheless, TRPM5 channel is a good candidate. It has been reported that TRPM5 channels are activated by lower concentrations of free intracellular calcium while high calcium levels result in inhibition of these channels resulting in a bell shaped dose response curve [START_REF] Prawitt | TRPM5 is a transient Ca 2+ -activated cation channel responding to rapid changes in [Ca 2+ ]i[END_REF]. We hypothesize that simultaneous administration of fat and bitter tasting agents results in very high increases in [Ca 2+ ]i due to the opening of store-operated calcium channels, following the activation of fat taste receptors. Such increases in [Ca 2+ ]i much higher than required, may trigger inhibition of TRPM5 channels and, consequently, inhibition of the bitter-taste perception. However, this hypothesis needs experimental confirmations.

Conclusions and perspectives

Our short, but comprehensive review, sheds light on the interaction of "bitter taste" and "bitter taste of lipids". However, there is a long way to better understand this interaction. The recent report on the involvement of olfaction in the perception of "fat bitterness" has twisted the story. In future, a number of behavioral and genetic studies are required to confirm this relationship in different populations of the world as by now only one study is available on this subject [START_REF] Barbarossa | Variant in a common odorant-binding protein gene is associated with bitter sensitivity in people[END_REF]. Besides, we have to explore the "cross-talk" between fat sensors like CD36 and GPR120 and olfactory receptors, both of nasal and buccal origins. Indeed, human taste bud cells have been found to express olfactory binding proteins (OBP) like OBPII (unpublished observations) which can via direct interaction, at the lingual papillae level, with CD36 or GPR120 might influence taste perception and feeding behavior. Furthermore, new studies are required to establish the implication of genetic polymorphism of OBIIa, CD36/GPR120 and TAS2R receptors in obesity. Nonetheless, we should not ignore the environmental/social factors, beside genetic polymorphism, in the incidence of obesity. Indeed, Burd et al. [START_REF] Burd | PROP taster status interacts with the built environment to influence children's food acceptance and body weight status[END_REF] have very elegantly shown that two children populations with TAS216 SNP were susceptible to develop obesity, and the one that was residing in a low socio-economic area developed more incidence of obesity than the one that was in high posh area. The exploration of a cross-talk between fat and bitter tastes might find applications even in areas other than food industry such as in the pharmaceutical industry as reducing the bitterness of the medicaments is highly desirable to make them acceptable for the patients, particularly for young ones.

Figure legends

Figure 1: Fatty acids bind to CD36 and GPR120 (1) while bitter molecules activate TAS2R receptors [START_REF] Roper | Taste buds: cells, signals and synapses[END_REF]. This binding results in the activation of PLCβ which hydrolyses PIP 2 to give rise to IP 3 [START_REF] Ozdener | CD36-and GPR120-mediated Ca 2+ signaling in human taste bud cells mediates differential responses to fatty acids and is altered in obese mice[END_REF]. Binding of IP 3 to IP 3 R on endoplasmic reticulum results in the release of calcium (4), which subsequently opens TRPM5 channels [START_REF] Mennella | The development of sweet taste: From biology to hedonics[END_REF]. An increase in the free intracellular concentrations of Na + following the opening of TRPM5 channels results in depolarization of the gustatory cell and the release of neurotransmitters, notably ATP and serotonin [START_REF] Murtaza | Alteration in taste perception in cancer: causes and strategies of treatment[END_REF]. Some bitter tastants like Quinine can bind directly and block potassium channels which in-turn results in depolarization and neurotransmitter release [START_REF] Roper | Taste buds: cells, signals and synapses[END_REF]. Notably, very high increases in [Ca 2+ ]i can inhibit TRPM5 channels which might explain the inhibition of bitter taste following the simultaneous administration of fat and bitter tastants. The interrogative marks (?) represent the possible direct interaction of K + channels and TRPM5 channels. [START_REF] Duffy | Food acceptance and genetic variation in taste[END_REF] Association between genetic variation in taste and acceptance for sweet, high-fat, and bitter foods and beverages n= (46, 24 female, 22 male; age (21± 6 years)

In women, liking for high-fat food decreased with increasing perceived bitterness of PROP. In men, liking for the same high-fat food increased but with increasing papillae densities.

Tepper and Nurse [START_REF] Tepper | PROP taster status is related to fat perception and preference[END_REF] Relationship between papillae density, bitter taste and fat taste perception Subjects were divided into 3 groups based on their sensitivity to PROP (n=25 for each group) Bitter non-tasters were not able to discriminate between low and high fat content in salad dressings. Fat perception and preference is related to genetic and anatomical differences among individuals.

Nasser et al. [22]

To assess whether PROP tasters are sensitive to the presence of bittertasting fatty acids n=37 (42 -43 years old);

The PROP tasters (7 males and 22 females); PROP nontasters (4 males and 4 females) PROP tasters correctly discriminated the sample containing the added free fatty acids.

Hayes and Duffy [START_REF] Hayes | Revisiting sugar-fat mixtures: sweetness and creaminess vary with phenotypic markers of oral sensation[END_REF] Investigation of the influence of PROP phenotype on fungiform papillae density and sweet and creamy sensations n=79 male subjects The fungiform papillae density generally showed similar effects as PROP on sweetness and creaminess.

Drewnowski et al. [START_REF] Drewnowski | Genetic sensitivity to 6-n-propylthiouracil has no influence on dietary patterns, body mass indexes, or plasma lipid profiles of women[END_REF] Associations between PROP responsiveness, food choices, and dietary intakes Female breast cancer patients (n=179) and cancer-free controls (n=179) PROP responsiveness was not associated with diets lower in fat, more favourable plasma lipid profiles, or with lower body mass indexes.

Mattes et al. [START_REF] Mattes | Effects of linoleic acid on sweet, sour, salty, and bitter taste thresholds and intensity ratings of adults[END_REF] Investigation of the sensitization of taste receptors by fatty-acids through inhibition of delayed-rectifying potassium channels PROP tasters (n=34, 17 male and 17 female) Presence of fatty acids reduced the perception of highest concentrations of caffeine, a bitter tast ant, but not sweet, sour or salty ratings.

Koriyama et al. [26]

To elucidate the effects of oils on taste perception using oil in water emulsions and different tastants A trained panel of 4 females and 5 males (25 to 45 years old) having previous experience in tasting emulsions Tuna oil suppressed bitterness but not sweetness or saltiness.

Pittman et al. [START_REF] Pittman | Linoleic and oleic acids alter the licking responses to sweet, salt, sour, and bitter tastants in rats[END_REF] Investigation of the licking response of rats to different taste modalities in the presence of fatty acids 38 naive male Sprague-Dawley rats greater than 90 days old Presence of fatty acids increased licking response to sweet taste solutions but inhibited for bitter tasting solution.

  Figure1

1 Table 1 :

 11 Summary of studies highlighting the interaction between fat and bitter taste perception

	A) Behavioural studies
	Study	Purpose	Sample	Findings
	Duffy	and
	Bartoshuk


Lorène J Lebrun

Jacques Grober
email: jacques.grober@agrosupdijon.fr

Could glucagon-like peptide-1 be a potential biomarker of early-stage intestinal ischemia?

Keywords: Intestinal ischemia, mesenteric ischemia, glucagon-like peptide-1, inflammation, lipopolysaccharides, biomarker

Introduction

Intestinal ischemia, also called mesenteric ischemia, is a severe gastrointestinal and vascular medical emergency caused by a sudden decrease or cessation of blood flow through the mesenteric vessels. Four types of vascular dysfunction can generate this condition: embolic arterial occlusion (40-50 %), thrombotic arterial occlusion (20-35 %), non-occlusive forms (5-15 %) and venous mesenteric thrombosis (5-15 %) [START_REF] Nuzzo | Management of mesenteric ischemia in the era of intestinal stroke centers: The gut and lifesaving strategy[END_REF]. Intestinal ischemia can originate from certain diseases (mainly atherosclerosis), trauma, shock, surgery or organ transplantation. It generates hypoperfusion of intestinal tissues and can rapidly progress to intestinal wall necrosis and infarction, local and systemic inflammation or even death if not treated in time.

The mortality of intestinal ischemia is still considerably high despite all the medical advances of the past few years, but improvements in outcome would require more rapid diagnosis and early appropriate medical care. Indeed, a prospective pilot study involving 18 patients has shown that early multimodal management of intestinal viability resulted in a significant reduction in intestinal necrosis and resection [START_REF] Gollin | Intestinal fatty acid binding protein in serum and urine reflects early ischemic injury to the small bowel[END_REF], the 2-year survival rate was 89% in these patients. One of the main issues concerning intestinal ischemia is the lack of specific, sensitive and early stage biomarkers to predict this condition prior to deadly complications.

The gut hormone glucagon-like peptide-1 (GLP-1) could be a promising early biomarker for intestinal ischemia. Originally described for its role in glucose metabolism, GLP-1 may have other functions, particularly in relation to gut inflammation [START_REF] Drucker | Mechanisms of Action and Therapeutic Application of Glucagon-like Peptide-1[END_REF][START_REF] Yusta | GLP-1R Agonists Modulate Enteric Immune Responses Through the Intestinal Intraepithelial Lymphocyte GLP-1R[END_REF]. Indeed, bacterial-derived pro-inflammatory lipopolysaccharides (LPS) are able to stimulate the secretion of GLP-1, and this phenomenon occurs rapidly after the onset of intestinal ischemia [START_REF] Lebrun | Enteroendocrine L Cells Sense LPS after Gut Barrier Injury to Enhance GLP-1 Secretion[END_REF][START_REF] Nguyen | Lipopolysaccharides-mediated increase in glucose-stimulated insulin secretion: involvement of the GLP-1 pathway[END_REF]. In this review, we first describe intestinal ischemia, its current and investigative diagnostic tools. We then present GLP-1 as a potential biomarker for the rapid diagnosis of intestinal ischemia, which would contribute to a reduction in mortality.

Intestinal ischemia

Pathophysiology

From an anatomical point of view, a large part of the gut (the entire small intestine and ascending colon) is vascularized by the superior mesenteric artery. It supplies the arterioles and the sub-mucosal capillary networks which irrigate the intestinal mucosa up to the tip of the villi. Figure 1 presents the pathophysiology of intestinal ischemia. Ischemic damage starts with desquamation and bleeding of mucosal villi, which is highly sensitive to hypoperfusion and hypoxia. The intestinal epithelium acts as a barrier against the translocation of luminal components. Rupture of this intestinal barrier rapidly triggers local and systemic inflammation with, respectively, the recruitment of inflammatory mediators and the passing of pro-inflammatory bacterial endotoxins and other microbial components into the bloodstream. This bacterial translocation can result in the development of systemic inflammation response syndrome (SIRS), which potentiates intestinal hypoperfusion and damage. The rapid reversal of ischemia through the reperfusion of digestive vessels is absolutely required to avoid irreversible necrosis and resection of intestinal tissue. However, and paradoxically, reperfusion of ischemic intestinal tissue may further aggravate the severity of the damage. Indeed, restoration of blood flow abruptly reintroduces oxygen that stimulates the production of free radicals, generates oxidative stress and accelerates necrosis [START_REF] Guan | Intestinal ischemia-reperfusion injury: reversible and irreversible damage imaged in vivo[END_REF]. This "vicious circle" can result in lethal intestinal ischemia-reperfusion complications with the dysfunction of additional organs. Intestinal ischemia is considered as a root cause of multiple organ failure (MOF) [START_REF] Harward | Multiple organ dysfunction after mesenteric artery revascularization[END_REF] and ultimately death which explains the urgency of the condition and the need for rapid medical care.

FIGURE 1: Pathophysiology of intestinal ischemia -reperfusion

Several pathophysiological and scalable steps occur after the onset of mesenteric ischemia. Some of them are reversible (above the dotted line), but disease progression is certain and irreversible once intestinal necrosis has begun (below the dotted line). On the one hand, vessel reperfusion is needed to counteract ischemia (green arrow), but it also generates oxidative stress which contributes to irreversible necrosis of tissues. Abbreviations: SIRS, systemic inflammatory response syndrome.

Epidemiology and current diagnostic tools

In large part because of the association with atherosclerosis, intestinal ischemia is commonly considered a disease that affects older people, with the typical age of onset being over 60 years [START_REF] Cardin | Clinical correlation of mesenteric vascular disease in older patients[END_REF]. However, because this pathology can occur in both sick and healthy vessels, cases have also been reported in younger people [START_REF] Nuzzo | Mo1300 Acute Mesenteric Ischemia in Young Patients: Epidemiological Study of 52 Cases[END_REF]. Despite significant medical advances, the high mortality for intestinal ischemia has not changed since the 1940s, and it concerns about 60% to 80% of patients [START_REF] Schoots | Systematic review of survival after acute mesenteric ischaemia according to disease aetiology[END_REF]. It is assumed that mesenteric ischemia represents 1/1000 hospitalizations worldwide [START_REF] Clair | Mesenteric Ischemia[END_REF], but this proportion is certainly underestimated [START_REF] Kärkkäinen | Acute Mesenteric Ischemia Is a More Common Cause than Expected of Acute Abdomen in the Elderly[END_REF] due to delays in diagnosis and treatment. The high mortality is also partially due to the difficulty of diagnosing early stage mesenteric ischemia. Indeed, a speedy and correct diagnosis is decisive for suitable medical care and the resulting patient prognoses. Before warning signs such as peritonitis appear, symptoms are unspecific (e.g. abdominal pain, nausea, vomiting) and conventional clinical markers such as serum lactate levels or white blood cells numbers are neither specific nor sensitive enough [START_REF] Derikx | Serological markers for human intestinal ischemia: A systematic review[END_REF]. Until now, there has been no biological marker that could diagnose intestinal ischemia at an early stage. Computerized tomography and angiography are radiological imaging techniques that may also be used and which are helpful diagnostic tests. However, they are costly, more invasive and can generate medical complications [START_REF] Glenister | Infarcted intestine: a diagnostic void[END_REF]. Most of all, it is difficult to manage radiological investigations in emergency settings and critical care medicine. Accordingly, there is an obvious need for specific and sensitive plasma biomarkers that would help clinicians to diagnose intestinal ischemia in the early stages.

Investigative plasma biomarkers

A recent study conducted in a specialized intestinal stroke centre demonstrated that, during intestinal ischemia, irreversible intestinal ischemic damage could be prevented or delayed through multidisciplinary management of the condition: preserving intestinal viability improves patient outcomes and survival [START_REF] Corcos | Effects of a multimodal management strategy for acute mesenteric ischemia on survival and intestinal failure[END_REF]. This development is definitely a great step forward for the medical care of intestinal ischemia, but rapid diagnosis is still needed. In the last few years, significant clinical and preclinical efforts have been made to find biomarkers which could predict gastrointestinal damage before it becomes irreversible and spreads to the rest of the body. D-lactate [START_REF] Demir | Beyond lactate: is there a role for serum lactate measurement in diagnosing acute mesenteric ischemia?[END_REF], intestinal fatty acid binding protein (I-FABP) [START_REF] Cronk | Intestinal fatty acid binding protein (I-FABP) for the detection of strangulated mechanical small bowel obstruction[END_REF], αglutathione S-transferase [START_REF] Khurana | Glutathione S-transferase: a potential new marker of intestinal ischemia[END_REF], albumin [START_REF] Dundar | Serum ischemia-modified albumin levels in an experimental acute mesenteric ischemia model[END_REF], D-dimer [START_REF] Chiu | D-dimer in patients with suspected acute mesenteric ischemia[END_REF], procalcitonin [START_REF] Cosse | Procalcitonin and intestinal ischemia: a review of the literature[END_REF], endothelin-1 [START_REF] Groesdonk | Elevated endothelin-1 level is a risk factor for nonocclusive mesenteric ischemia[END_REF] or smooth muscle protein 22 [START_REF] Schellekens | SM22 a Plasma Biomarker for Human Transmural Intestinal Ischemia[END_REF] have been studied as potential biomarkers for mesenteric ischemia. Some are certainly promising while others lack specificity, sensitivity or rapidity, but all require in depth study to assess their diagnostic accuracy [START_REF] Derikx | Serological markers for human intestinal ischemia: A systematic review[END_REF].

GLP-1: a promising early biomarker

Glucagon-like peptide-1 (GLP-1)

GLP-1 is a hormone produced by enteroendocrine L cells which are found primarily in the epithelium of the distal ileum and colon. These cells express proglucagon as well as prohormone convertase 1/3, a specific peptidase able to cleave proglucagon into several peptides including GLP-1. For some years now, GLP-1 has generated great clinical interest for the treatment of metabolic disorders such as type 2 diabetes because of its role in the regulation of glucose homeostasis [START_REF] Drucker | Mechanisms of Action and Therapeutic Application of Glucagon-like Peptide-1[END_REF]. Initially described for its incretin function, and hence its insulinotropic role in pancreatic beta cells [START_REF] Kreymann | Glucagon-like peptide-1 7-36: a physiological incretin in man[END_REF], GLP-1 has subsequently been shown to have many other functions in glucose metabolism. Indeed, this gut-derived hormone is also able to stimulate insulin biosynthesis [START_REF] Drucker | Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line[END_REF], inhibit glucagon production [START_REF] Kreymann | Glucagon-like peptide-1 7-36: a physiological incretin in man[END_REF], delay gastric emptying [START_REF] Nauck | Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans[END_REF] or even induce satiety and reduce food intake [START_REF] Turton | A role for glucagon-like peptide-1 in the central regulation of feeding[END_REF]. Secretion of GLP-1 from intestinal L cells is mostly triggered by ingested nutrients and involves various and intricate molecular mechanisms [START_REF] Lim | Glucagon-Like Peptide 1 Secretion by the L-Cell: The View From Within[END_REF][START_REF] Tian | The incretin hormone GLP-1 and mechanisms underlying its secretion[END_REF]. Carbohydrates, lipids and proteins are all able to stimulate GLP-1 secretion through direct interaction with their specific membrane receptors on the surface of L cells (Figure 2). However, the densest population of L cells is located in the distal gut, and postprandial plasma GLP-1 levels increase rapidly, even before nutrients reach this part of the gut [START_REF] Borgstrom | Studies of intestinal digestion and absorption in the human[END_REF]. This physiological discrepancy suggests the implication of other secretion mechanisms, and, accordingly, the existence of hormonal and neural secretion pathways has been previously studied. Overall, GLP-1 secretion can be activated by a wide range of L cell membrane receptors that recognize nutrients but also molecules such as neurotransmitters, hormones or even inflammatory compounds (Figure 2).

FIGURE 2: Cellular signalling pathways responsible for GLP-1 secretion

GLP-1 secretion from enteroendocrine L cells can be stimulated by different factors (nervous, protein, inflammatory…). Each of these pathways involves various membrane receptors. Some of them are well-described, some are still controversial and some are completely unknown yet (polyphenols and IL-6). Abbreviations: TLR4, toll-like receptor 4; IL-6, interleukin 6; GLP-1, glucagonlike peptide-1; GLUT, glucose transporter; SGLT1, sodium glucose linked transporter 1; T1R, type 1 taste G-protein coupled receptors; PEPT1, peptide transporter 1; GPRC6A, G protein-coupled receptor family c group 6 subtype A; GPR, G protein coupled receptor; FATP4, fatty-acid binding protein 4; InsR, insulin receptor; GLP-1R, GLP-1 receptor; Ob-Rb, leptin receptor; SSTR5, somatostatin receptor type 5; GIPR, glucose-dependent insulinotropic polypeptide receptor; TGR5, membranetype receptor for bile acids; GPRR, gastrin-releasing peptide receptor; GABA A , γ-aminobutyric acid receptor; GlycinR, glycin receptor; PK, protein kinase; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; cAMP, cyclic adenosine monophosphate; Epac2, exchange protein activated by cAMP 2.

GLP-1, lipopolysaccharides and inflammation

Besides its originally described glucoregulatory role, GLP-1 has also more recently been investigated for its anti-inflammatory properties and its link with sepsis and critical illness.

Previous studies have demonstrated that the endogenous GLP-1 system is activated during sepsis [START_REF] Perl | Sepsis-induced activation of endogenous GLP-1 system is enhanced in type 2 diabetes[END_REF], have associated GLP-1 levels with predicted mortality [START_REF] Perl | Sepsis-induced activation of endogenous GLP-1 system is enhanced in type 2 diabetes[END_REF][START_REF] Lebherz | GLP-1 Levels Predict Mortality in Patients with Critical Illness as Well as End-Stage Renal Disease[END_REF] and have correlated increased levels of circulating GLP-1 with the extent of concomitant critical illness [START_REF] Lebherz | GLP-1 Levels Predict Mortality in Patients with Critical Illness as Well as End-Stage Renal Disease[END_REF][START_REF] Ingels | Glucose homeostasis, nutrition and infections during critical illness[END_REF][START_REF] Kahles | GLP-1 secretion is increased by inflammatory stimuli in an IL-6-dependent manner, leading to hyperinsulinemia and blood glucose lowering[END_REF]. GLP-1 may exert both distant and local beneficial anti-inflammatory effects. For instance, it has been described as protective for the heart, lung or liver, and some studies highlight a role for GLP-1 in maintaining gut homeostasis [START_REF] Drucker | Mechanisms of Action and Therapeutic Application of Glucagon-like Peptide-1[END_REF][START_REF] Yusta | GLP-1R Agonists Modulate Enteric Immune Responses Through the Intestinal Intraepithelial Lymphocyte GLP-1R[END_REF]. GLP-1 based drugs, initially studied and used in the context of glucose homeostasis, are now investigated in the treatment of various inflammatory disorders (gallego-colon 2018, Drucker 2018, Insuela 2017). This potential anti-inflammatory role of GLP-1 is in line with its recently described inflammatory-induced secretion. LPS are pro-inflammatory molecules found at the surface of Gram negative bacteria and in huge amounts within the gut microbiota. In vivo, LPS were shown to induce glucose-stimulated insulin secretion through an increase in GLP-1 plasma levels. The insulinotropic effect of LPS was strongly diminished when mice were pretreated with a GLP-1 receptor antagonist or when GLP-1 receptor deficient mice were used [START_REF] Nguyen | Lipopolysaccharides-mediated increase in glucose-stimulated insulin secretion: involvement of the GLP-1 pathway[END_REF].

Among the inflammatory mediators that have been shown to increase GLP-1 plasma levels, pro-inflammatory cytokine IL-6 increases insulin secretion in vivo through the stimulation of GLP-1 synthesis and secretion in the intestines [START_REF] Ellingsgaard | Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells[END_REF]. Moreover, it was recently shown that LPS molecules are able to directly stimulate GLP-1 secretion through activation of the tolllike receptor 4 (TLR4) of enteroendocrine L cells [START_REF] Lebrun | Enteroendocrine L Cells Sense LPS after Gut Barrier Injury to Enhance GLP-1 Secretion[END_REF].

GLP-1 as a biomarker of intestinal ischemia

In inflammatory settings, intestinal ischemia is associated with a prompt and significant increase in GLP-1 plasma levels. As soon as intestinal barrier begins to disorganize, which is right after the onset of experimental mesenteric ischemia in mice, luminal LPS (and certainly other molecules) have more access to epithelial enteroendocrine L cells and can trigger GLP-1 secretion. The LPS / GLP-1 cascade, first described in mice [START_REF] Lebrun | Enteroendocrine L Cells Sense LPS after Gut Barrier Injury to Enhance GLP-1 Secretion[END_REF][START_REF] Nguyen | Lipopolysaccharides-mediated increase in glucose-stimulated insulin secretion: involvement of the GLP-1 pathway[END_REF][START_REF] Kahles | GLP-1 secretion is increased by inflammatory stimuli in an IL-6-dependent manner, leading to hyperinsulinemia and blood glucose lowering[END_REF], works similarly in humans. Injection of a low dose of LPS led to a significant rise in GLP-1 plasma levels in healthy volunteers, and GLP-1 secretion was rose rapidly in an experimental model of intestinal ischemia in humans [START_REF] Lebrun | Enteroendocrine L Cells Sense LPS after Gut Barrier Injury to Enhance GLP-1 Secretion[END_REF]. These results suggest that GLP-1 could be a promising biomarker for early stage intestinal ischemia which could help to identify the condition in the critical first, reversible steps. Unlike other promising biomarkers which are released only after intestinal cell death (e.g. I-FABP) or systemically generalized inflammation (e.g. Dlactate or procalcitonin), the increase in GLP-1 secretion occurs earlier and is the physiological response of functional enteroendocrine L cells to the luminal compounds which are usually kept at a distance. GLP-1 secretion is therefore quicker and more sensitive, making GLP-1 a potential biomarker for intestinal ischemia and opening new perspectives in diagnosis and medical care of this fatal illness. GLP-1 meets all the criteria necessary to be an optimal marker of intestinal pain and distress: 1) it is produced by mucosal intestinal cells, 2) hepatic proteolytic activity is insignificant since GLP-1 is quantified under its total (active and inactive) form, and 3) it is detectable and quantifiable in systemic circulation. This discovery could counter the cruel lack of clinical biomarkers available to diagnose and therefore manage intestinal ischemia efficiently in the early stages. GLP-1 could thus become part of a panel of biomarkers for intestinal ischemia and could help to reduce the associated high mortality rates. In addition, GLP-1 testing could become a routine laboratory test provided as part of the ELISA kit already on the market.

Conclusion

A large number of preclinical and clinical studies aim to improve the diagnosis and outcomes of intestinal ischemia through identification of specific and sensitive biomarkers which are able to predict the disease in the early stages. Here, the gut-derived hormone GLP-1 is described as a potential early biomarker of this severe condition: GLP-1 plasma levels rise rapidly in both mice and humans with intestinal ischemia. In the context of clinical care, further work is needed to 1) assess whether GLP-1 levels reflect the severity of gastrointestinal damage in humans and 2) confirm whether GLP-1 can efficiently diagnose intestinal ischemia. Since GLP-1 is not clinically used to diagnose any pathology so far, future clinical investigations are also needed to determine a reference plasma level for GLP-1 which would be used for comparison purposes. Indeed, GLP-1 levels can vary inter-and intraindividually (fasting or postprandial state). However, consistently with our preliminary results described here, we assume that the pathological increase in GLP-1 levels would be greater than the physiological one and therefore easily differentiated. Finally, from a fundamental research point of view, it would also be interesting to investigate the role of GLP-1 secretion after intestinal ischemia. After the discovery of GLP-1's anti-inflammatory properties, we could assume that it promotes the restoration of mucosa and reduces inflammation.

Acknowledgements

This work was supported by the INSERM, Centre de Recherches UMR 1231, the University Bourgogne Franche-Comte, a French government grant managed by the French National Research Agency under the program ''Investissements d'Avenir'' with reference ANR-11 LABX-0021, the FEDER, and the Regional Council of Bourgogne Franche-Comte. The authors gratefully acknowledge S. Rankin for English review and P. Mourey for graphical help.

Author contributions

Writing -original draft and visualization, L.J.L. and J.G.; writing -review & editing, L.J.L. and J.G.; supervision, J.G.; declarations of interest, none.