# LEAP2: from feeding regulation to its implications in eating disorders

Francesca Mariuz, Tatiana Landré, Chloé Tezenas Du Montcel, Alexandre

Benani, Thierry Bienvenu, Virginie Tolle

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Francesca Mariuz, Tatiana Landré, Chloé Tezenas Du Montcel, Alexandre Benani, Thierry Bienvenu, et al.. LEAP2: from feeding regulation to its implications in eating disorders. Physiology & behavior, 2025, 300, pp.115013. 10.1016/j.physbeh.2025.115013. hal-05200498

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Submitted on 5 Aug 2025

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# LEAP2: from feeding regulation to its implications in eating disorders

Francesca Mariuz<sup>a</sup>, Tatiana Landré<sup>b</sup>, Chloé Tezenas du Montcel<sup>a,c</sup>, Alexandre Benani<sup>b</sup>, Thierry Bienvenu<sup>a</sup>, Virginie Tolle<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Ghrelin

LEAP2

GHSR

Feeding

Eating disorders

Genetic variants

## ABSTRACT

Almost twenty years after the discovery of ghrelin, a potent Growth Hormone Secretagogue (GHS) and orexigenic peptide synthesized in the stomach and acting on its cognate receptor, the GHSR, LEAP2 (Liver-Expressed Antimicrobial Peptide 2), produced mainly in the liver and small intestine, was identified as an endogenous ghrelin antagonist. This article reviews the initial characterization of LEAP2 as an antimicrobial peptide and its role in opposing ghrelin's actions on food intake, GH secretion, hedonic feeding and energy homeostasis. We also describe the long-term regulation of ghrelin and LEAP2 by positive and negative energy balance, and their short-term regulation by meals and nutrient intake. We then discuss the effect of LEAP2 variants on the concentration and function of LEAP2 and their possible associations with altered eating behaviors. Finally, the potential implications of LEAP2 in obesity or pathologies associated with abnormal feeding behavior such as anorexia nervosa are also highlighted. Due to their opposite actions on food intake, energy homeostasis, glucose homeostasis, hedonic feeding and reward, ghrelin and LEAP2 represent key pharmacological targets for disorders impacting energy homeostasis and eating. These discoveries open up new perspectives for the understanding of the mechanisms that regulate eating behaviors and more broadly energy balance, relayed by the GHSR, in physiological and pathological conditions.

## 1. Introduction: discovery, structure and function of LEAP2 as an anti-microbial peptide and endogenous ghrelin antagonist

The discovery of LEAP2 (Liver-expressed antimicrobial peptide 2) dates back to 2002, when it was first isolated from a large-scale human blood ultrafiltrate peptide library [1]. *In vitro*, LEAP2 has antimicrobial properties against the growth of Gram-positive, but not Gram-negative, bacteria at concentrations in the micromolar range and exhibits dose-dependent antimicrobial activity against several microbial model organisms in a variety of species [2,3]. The antibacterial role of LEAP2 in humans has not been extensively studied, possibly due to the fact that the concentration required for the peptide to have an antimicrobial effect is 3000 times higher compared to the physiological range commonly observed in human plasma [4]. Nevertheless, LEAP2 may be physiologically involved in immunological processes, as suggested by the fact that its concentration in the cerebrospinal fluid (CSF) of patients affected by bacterial meningitis is significantly increased, and this is

reversed by antibiotic treatment. In addition, blood levels of LEAP2 are increased in people suffering from bacteremia [5].

LEAP2, which belongs to the liver-expressed antimicrobial peptide (LEAP) family, is a cysteine-rich and cationic protein with a core structure stabilized by two disulfide bridges formed by cysteine residues in the 1-3 and 2-4 relative positions. The strong basicity and the two disulfide bonds of LEAP2 facilitate its penetration into the bacterial membrane and give LEAP2 its bactericidal activity [3]. Synthesized as a 77- or 76-residue precursor in human and mouse, respectively, pre-proLEAP2 is predominantly expressed by hepatocytes and enterocytes [6]. Despite having a highly conserved structure, LEAP2 expression patterns differ among mammals. In particular, in humans and rats it is predominantly expressed in the liver [1,7], while in mice the highest expression levels are observed in the small intestine (highest in jejunum, followed by duodenum and ileum), where LEAP2 is specifically found in enterocytes along the epithelial layer [6]. LEAP2 expression has also been measured in the human brain and transcripts were found to be

<footnote><sup>a</sup> Université Paris Cité, Institute of Psychiatry and Neuroscience of Paris, INSERM U1266, Team "Vulnerability to Psychiatric and Addictive Disorders", F-75014 Paris, France</footnote>

<footnote><sup>b</sup> Centre des Sciences du Goût et de l'Alimentation, CNRS, INRAE, Institut Agro Dijon, Université Bourgogne Europe, Team Neurobiology of feeding behaviors, Dijon, France</footnote>

<footnote><sup>c</sup> Metabolism and Nutrition Research Group, Louvain Drug Research Institute (LDRI), UCLouvain, Université Catholique de Louvain, Brussels, Belgium.</footnote>

<footnote>* Corresponding author at: Institute of Psychiatry and Neuroscience of Paris, INSERM U1266, 102-108 rue de la Santé, 75014 Paris, France.
E-mail address: virginie.tolle@inserm.fr (V. Tolle).</footnote>
either at extremely low levels or absent [1,8] while in rodent brains LEAP2 mRNA were undetectable [6,7].

The largest form of LEAP2 contains 40 amino-acid residues. This mature peptide is generated from a precursor after processing by a signal peptidase at a putative cleavage site for a furin-like endoprotease. Mature LEAP2 contains a N-terminal hydrophobic domain, a cationic core region, and a C-terminal domain. In contrast to smaller LEAP2 variants, this 40 amino-acid peptide exhibits dose-dependent antimicrobial activity [1].

Interestingly, early on it was suggested that LEAP2 could exhibit other fundamental functions beyond its antimicrobial properties, explaining its highly preserved sequence among vertebrates [9]. In 2018, about 15 years after its initial discovery, LEAP2 was identified as an endogenous antagonist of the Growth Hormone Secretagogue Receptor 1a (GHSR1a) by screening this peptide against a panel of 168 engineered stable G-protein-coupled receptor (GPCR)-expressing cell lines [6]. Ghrelin is a 28 amino-acid acylated peptide produced mainly in the stomach and originally isolated as the endogenous ligand of the GHSR and for its potent growth hormone (GH) secretagogue activity [11]. It was independently identified by another group due to its similarities with another gastrointestinal peptide, motilin [12]. It was further characterized as a powerful orexigenic peptide [13,14]. Ghrelin also exists in a non acylated form that does not bind to GHSR and which function is less well characterized [10]. We will use the term “ghrelin” when referring to the acylated peptide.

In vitro studies highlighted that physiological concentrations of LEAP2 could fully inhibit ghrelin-induced GHSR activation in a non-competitive manner, possibly by acting as an allosteric modulator of the receptor [6]. In vivo experiments allowed to better characterize the effects of LEAP2 on GHSR- and ghrelin-dependent processes. One of the most well-known functions of ghrelin is its ability to stimulate food intake [15]. Subcutaneous (sc) injection of LEAP2 abolishes ghrelin-induced food intake in mice, and this effect has been reproduced in multiple studies using different concentrations and routes of administration of the two peptides (Table 1) [6,7,16,17]. Thus, LEAP2 appears to operate, at least partly, as an “anti-ghrelin” hormone, counteracting the orexigenic effects of ghrelin. LEAP2 was originally thought to be a

non-competitive antagonist at the GHSR (i.e. inhibits the action of ghrelin by binding to a different site on the receptor). However, subsequent pharmacological studies have shown that LEAP2 actually competes for binding sites with ghrelin (i.e. acts a competitive antagonist). In addition, LEAP2 has also an inverse agonist activity in the absence of ghrelin, maintaining GHSR in an inactive conformation and blocking its constitutive activity, as demonstrated both *in vitro* and *ex vivo* [18,19]. For example, LEAP2 affects the basal level of inositol phosphate 1 production in HEK293T cells expressing GHSR [18], an effect for which the N-terminal portion of the peptide is sufficient, and impairs the reduction of Cav2.2 currents induced by constitutive GHSR activities [20]. In addition, whole-cell patch-clamp recordings in brain slices showed that LEAP2 hyperpolarizes Neuropeptide Y (NPY) neurons, which highly express GHSR [19]. Finally, LEAP2 reduces the ability of GHSR to form heterodimers with other receptors, such as the dopamine receptor D2 (DRD2) *in vitro* [20].

## 2. Role of LEAP2 in the regulation of energy balance and homeostatic food intake

### 2.1. Role of LEAP2 in the regulation of food intake and energy homeostasis

As aforementioned, ghrelin injection in mice pre-treated with LEAP2 has been shown to impair ghrelin-induced food intake in several studies [6,7,16,17]. Of note, intraperitoneal (ip) injection of LEAP2 followed by intracerebroventricular (icv) injection of ghrelin does not affect food intake, whereas icv injection of LEAP2 followed by ip administration of ghrelin does, suggesting that the kinetics of access of ghrelin and LEAP2 to GHSR is important for their actions as competitive antagonists [7]. Consistent with the antagonistic effect of LEAP2 on ghrelin-induced food intake, the dose of ghrelin required to induce food intake in whole-body LEAP2 knock-out (KO) mice is reduced by half compared to wild-type mice [21]. Structurally, the fundamental part of LEAP2 to antagonize GHSR signalling is its N-terminal domain, which stabilizes GHSR in an inactive conformation [18,22]. Indeed, this N-terminal domain is sufficient to mediate the anorexigenic effect of LEAP2, as demonstrated by

<table><caption>**Table 1**
Effects of ghrelin and LEAP2 co-administration on feeding, GH secretion and cFos activation.</caption><thead><tr><th>Peptides used</th><th>LEAP2 dose and route</th><th>Ghrelin dose and route</th><th>Treatment interval</th><th>Effects</th><th>Model</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="3">LEAP2 (1–40), Ghrelin</td><td>7,2 nmol/kg, ip</td><td>6 nmol/kg, iv</td><td>10 min</td><td>Decreased ghrelin-induced GH release</td><td>2–3 months old male C57BL/6 mice</td><td>[6]</td></tr><tr><td>3 µmol/kg, sc</td><td>0,15 µmol/kg, sc</td><td>10 min</td><td>Inhibition of ghrelin-induced food intake</td><td></td><td></td></tr><tr><td>0,15 µmol/kg, sc</td><td>0,15 µmol/kg, sc</td><td>10 min</td><td>No effect on ghrelin-induced food intake</td><td></td><td></td></tr><tr><td>LEAP2 (1–40), Ghrelin</td><td>10 pmol, icv</td><td>60 pmol, icv</td><td>0 min</td><td>Inhibition of ghrelin-induced food intake</td><td>8–12 weeks old male C57BL/6 J mice</td><td>[22]</td></tr><tr><td rowspan="3">LEAP2 (1–40), Ghrelin</td><td>1 nmol, icv</td><td>0,1 nmol, icv</td><td>Co-administration</td><td>Inhibition of ghrelin-induced food intake, cFos induction in ArcN and PVH and blood glucose</td><td>8 weeks old Wistar rats</td><td>[7]</td></tr><tr><td>15 nmol, ip</td><td>5 nmol, ip</td><td>30 min</td><td>Inhibition of ghrelin-induced food intake, cFos induction in ArcN and PVH and blood glucose</td><td></td><td></td></tr><tr><td>15 nmol, ip</td><td>0,1 nmol, icv</td><td>30 min</td><td>No effect on ghrelin-induced food intake</td><td></td><td></td></tr><tr><td>1 nmol, icv</td><td>5 nmol, ip</td><td>30 min</td><td>Inhibition of ghrelin-induced food intake, cFos induction in ArcN and PVH and blood glucose</td><td></td><td></td></tr><tr><td>LEAP2 (1–40), Ghrelin</td><td>1,5 nmol icv</td><td>0,45 nmol, icv</td><td>10 min</td><td>Inhibition of ghrelin-induced food intake</td><td>C57BL/6 male mice</td><td>[25]</td></tr><tr><td rowspan="2">LEAP2 (1–40), Ghrelin</td><td>8 nmol icv</td><td>1,5 nmol, icv</td><td>10 min</td><td>Inhibition of ghrelin-induced food intake</td><td>Sprague-Dawley rats</td><td>[17]</td></tr><tr><td>0,6 µmol/kg, ip</td><td>0,06 µmol/kg, ip</td><td>NA</td><td>Inhibition of ghrelin-induced food intake</td><td>12 weeks old C57BL/6 J mice</td><td>[17]</td></tr><tr><td>Palmitoylated LEAP2 (1–14), [Dpr3]Ghrelin</td><td>2 µmol/kg, sc</td><td>1 mg/kg, MW NA, sc</td><td>15 min</td><td>Inhibition of ghrelin-induced food intake and GH release</td><td></td><td>[23]</td></tr><tr><td>LEAP2 (1–14), [Dpr3] Ghrelin</td><td>1 µmol/kg, sc</td><td>1 mg/kg, MW NA, sc</td><td>15 min</td><td>Inhibition of ghrelin-induced food intake and GH release</td><td></td><td></td></tr><tr><td>LEAP2 (1–40), Ghrelin</td><td>150 pmol icv</td><td>150 pmol, icv</td><td>30 min</td><td>Inhibition of ghrelin-induced food intake</td><td>Adult male Sprague-Dawley rats</td><td>[27]</td></tr><tr><td>LEAP2 (1–40), Ghrelin</td><td>1 and 3 nmol icv</td><td>0,1 nmol, icv</td><td>NA</td><td>Decrease of ghrelin-induced food intake</td><td>3 weeks old male Sprague-Dawley rats</td><td>[24]</td></tr></tbody></table>
<table><caption>**Table 2**
Effects of LEAP2 acute and chronic administrations on food intake and metabolic parameters.</caption><thead><tr><th>Peptide and experimental conditions</th><th>Dose and route</th><th>Effects</th><th>Model</th><th>Reference</th></tr></thead><tbody><tr><td><strong>Acute treatment</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>LEAP2(1–40) at the onset of the dark phase</td><td>1 nmol, icv</td><td>No effect on food intake</td><td>8 weeks old<br>Wistar rats</td><td>[7]</td></tr><tr><td>LEAP2(1–40) after an overnight fast</td><td>0,45 and 1,5 nmol, icv</td><td>No effect on rebound feeding</td><td>C57BL/6 male mice</td><td>[25]</td></tr><tr><td>LEAP2(1–40) after a 4 h fast, onset of the dark phase</td><td>0,45 and 1,5 nmol, icv</td><td>Decreased rebound feeding</td><td>Sprague-Dawley rats</td><td>[49]</td></tr><tr><td>LEAP2(1–40)</td><td>3 µmol/kg, sc</td><td>Decrease in food intake and blood glucose</td><td>C57BL/6 J mice</td><td>[49]</td></tr><tr><td>LEAP2(1–40) after a 24 h fast</td><td>0,6 µmol/kg, ip</td><td>No effect on food intake</td><td>12 weeks old</td><td>[17]</td></tr><tr><td>LEAP2(1–40) after a 4 h fast, onset of the dark phase</td><td>10–100 nmol, icv</td><td>No effect on food intake</td><td>C57BL/6 J mice</td><td>[26]</td></tr><tr><td>Palmitoylated LEAP2(1–14) after a 17 h fast</td><td>2 µmol/kg, sc</td><td>Decreased food intake, increased cFos staining in POMC neurons</td><td>8 weeks old<br>C57BL/6 J male mice</td><td>[23]</td></tr><tr><td>LEAP2(1–14) after a 17 h fast</td><td>1 µmol/kg, sc</td><td>Highly decreased rebound feeding</td><td>12 weeks old</td><td>[23]</td></tr><tr><td>LEAP2(1–40), onset of the dark phase</td><td>0150 and 1,5 nmol, icv</td><td>Decreased rebound feeding</td><td>C57BL/6 J males</td><td>[27]</td></tr><tr><td>LEAP2(1–40), light phase</td><td>3 nmol, icv</td><td>Decreased night time feeding and body weight after 24 h</td><td>Adult Sprague-Dawley male rats</td><td>[24]</td></tr><tr><td><strong>Chronic treatment</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Subcutaneous pump, LEAP2(1–40) icv for 6 days</td><td>2,18 nmol/day, sc</td><td>No difference in food intake or body weight</td><td>C57BL/6 J mice</td><td>[17]</td></tr><tr><td>Use of subcutaneous osmotic pump to deliver LEAP2(1–40) icv for 6 days in mice food restricted to 40 % of their ad libitum food intake</td><td>2,18 nmol/day, sc</td><td>Significant decrease in body weight, blood glucose and liver weight, upregulation of IL-6 and IL-1β in the liver</td><td></td><td></td></tr><tr><td>Daily injections of LEAP2(1–40) at the onset of the dark phase</td><td>10 nmol/day, icv</td><td>Hypophagia and weight loss</td><td>8 weeks old<br>C57BL/6 J male mice</td><td>[26]</td></tr><tr><td>Use of osmotic pump to deliver LEAP2(1–40) icv for 7 days</td><td>2 nmol/day, icv</td><td>Decrease in food intake and body weight, circulating triglycerides, ACC in adipose tissue and increase of UCP1 in BAT</td><td>C57BL/6 J mice</td><td>[16]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Peptide and experimental conditions</th><th>Dose and route</th><th>Effects</th><th>Model</th><th>Reference</th></tr></thead><tbody><tr><td>Osmotic pump to deliver LEAP2(1–40) icv for 7 days after 12 weeks of HFD</td><td>2 nmol/day, icv</td><td>= food intake, body weight and metabolic parameters</td><td>DIO C57BL/6 J mice</td><td>[16]</td></tr><tr><td>Palmitoylated LEAP2(1–14) twice a day for two weeks after 4 months of HFD</td><td>2 µmol/kg, sc</td><td>= food intake, body weight and metabolic parameters</td><td>DIO C57BL/6 N male mice</td><td>[75]</td></tr><tr><td>Palmitoylated LEAP2(1–14) twice a day from the beginning of HFD</td><td>2 µmol/kg, sc</td><td>Decreased food intake and body weight, liver weight, leptin levels and de novo lipogenesis</td><td>C57BL/6 N male mice</td><td>[75]</td></tr></tbody></table>

the fact that LEAP2 (1–14) antagonizes both ghrelin- and [Dpr³] ghrelin-induced food intake [22,23]. Furthermore, palmitoylated LEAP2 (1–14), an analogue with a lipid modification that increases the stability of the peptide, shows an even stronger effect, possibly due to its higher affinity for GHSR and its increased half-life [23].

Several studies have attempted to dissect the effect of LEAP2 on food intake under more physiological conditions as well (Table 2). When administered during the light phase, LEAP2 reduces food intake in both mice and rats [6,24,25]. However, whereas in mice administration at the onset of the dark phase requires significantly higher concentrations than those required to suppress feeding during the light cycle (at least 10 nmol injected directly into the arcuate nucleus) [26], icv 4th ventricle injection in the range of 1.5 nmol is sufficient for rats [27]. The effect of LEAP2 on *ad libitum* food intake has also been studied in humans: LEAP2 concentrations were negatively correlated with preprandial hunger sensations assessed by Visual Analog Scores (VAS) in both normal weight, overweight and obese subjects [28]. Consistent with this, another study found an association between postprandial plasma LEAP2 and suppression of eating and a decrease in appetite ratings. This was associated with a reduction in responsiveness to food cues for both high and low energy foods in several brain regions, as measured by blood-oxygenation level dependent functional magnetic resonance imaging (BOLD fMRI) [29].

The chronic effects of LEAP2 have also been investigated: while chronic peripheral infusion of LEAP2 achieved via sc implantation of osmotic pumps had no effect on *ad libitum* food intake [17], chronic icv delivery via osmotic pumps led to a significant reduction in both food intake and body weight over seven days [16]. The discrepancy between these two reports may be due to the fact that in the former, osmotic pumps delivered 2.18 nmol of the peptide per day subcutaneously, whereas in the latter 2 nmol per day were delivered, resulting in a higher amount of the peptide in the brain.

The effect of LEAP2 on food intake has also been studied under conditions where ghrelin levels are physiologically elevated, such as in response to fasting. LEAP2 icv injection was shown to significantly reduce rebound food intake following an overnight fast in rats but not in mice fed a standard diet, possibly because the latter deplete their energy stores earlier [25]. However, subcutaneous injection of the more stable palmitoylated analogue of LEAP2 was able to significantly reduce rebound feeding for over 7 h in 17 h-fasted male mice [23]. These results suggest that LEAP2 may also act by antagonizing high levels of endogenous ghrelin, although it is not excluded that the effect of LEAP2 in fasted conditions might be independent of ghrelin, since rebound feeding and activation of paraventricular corticotropin-releasing hormone (CRH) neurons after fasting have previously been demonstrated to depend, at least partly, on GHSR constitutive activity [30,31].

## 2.2. Role of LEAP2 in the modulation of circuits involved in homeostatic feeding behaviour

One of the key neurobiological substrates of ghrelin-induced food intake is the arcuate nucleus (ArcN), which mediates whole-body energy homeostasis through the activity of Agouti Related Protein (AgRP)/ Neuropeptide Y (NPY) neurons and proopiomelanocortin (POMC) neurons, two neuronal populations with opposing roles in modulating food intake, exerting respectively orexigenic and anorexigenic effects. Under conditions of energy deficit, ghrelin binds to GHSR, which is expressed by NPY/AgRP neurons, leading to their activation and the release of AgRP, NPY and γ-aminobutyric acid (GABA) which is co-expressed in NPY/AgRP neurons. This promotes feeding in two ways: NPY and AgRP signalling will lead to the activation, either directly or indirectly, of both intra- and extra-hypothalamic nuclei involved in feeding induction, while GABA will inhibit POMC neurons, blocking their anorexigenic effect [32,33]. *Ex vivo* whole-cell patch-clamp recordings of NPY-GFP neurons showed that LEAP2 treatment hyperpolarizes this neuronal population and was even able to reverse the ghrelin-induced depolarization of their membrane [19]. The use of fluorescently labelled LEAP2 showed that the peptide, when administered icv in mice, is indeed able to bind to GHSR in the ArcN [22]. Co-administration of LEAP2 and NPY did not elicit any effect on food intake [7,25], further confirming that LEAP2 acts upstream of GHSR signalling. Ghrelin administration is known to induce cFos activation in the ArcN, but this effect is abolished by the co-administration of LEAP2 [7]. Consistently, ghrelin administration in LEAP2 knock-out mice leads to a significantly higher induction of cFos in the ArcN [21]. While icv administration of LEAP2 alone has no effect on ArcN cFos induction in *ad libitum* fed mice [31], chronic 3-days peripheral delivery of the N-terminal LEAP2 analogue, LEAP2(1–12), mediated by osmotic pumps results in a significant decrease in ArcN cFos staining after a 2-days fast [31]. Consistently, overexpression of LEAP2 in the arcuate nucleus (ArcN) using adeno-associated viral vectors resulted in a significant decrease in food intake and body weight [26].

Overall, these findings suggest that LEAP2 is able to inhibit the GHSR-mediated activation of NPY/AgRP neurons. LEAP2 also exerts an effect on POMC neurons: icv delivery of LEAP2 has been shown to induce cFos activation in this neuronal population, and this correlates with increased levels of *Pomc* mRNA. Similarly, LEAP2 treatment increases the activity of POMC neurons *ex vivo* measured using the calcium sensor GCAMP, and chemogenetic inhibition of POMC neurons blocks LEAP2-induced feeding suppression [26] (Fig. 1).

In addition to its well-known actions on the hypothalamic ArcN, ghrelin has multiple sites where its orexigenic action is mediated. The caudal brainstem has been shown to mediate ghrelin's orexigenic effect but with distinct pathways than forebrain ghrelin administration,
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Effects of LEAP2 on energy homeostasis, GH axis activity, reward processes and LEAP2 regulation by energy status, meals and nutrients. In the ArcN, GHSR is largely expressed on NPY/AgRP neurons and in a smaller proportion in POMC neurons. LEAP2 is participating in the control of the whole-body energy homeostasis by inhibiting the orexigenic effect of NPY/AgRP neurons and by activating POMC neurons, thus opposing ghrelin's actions. By modulating the activity of the GH axis, LEAP2 prevents ghrelin-induced Growth Hormone (GH) secretion and in calorie-restricted conditions reduces blood glucose, an effect that could be due to the lack of anti-hypoglycaemic action of GH, a hormone known to be involved in glycogenolysis and gluconeogenesis. In the Ventral Tegmental Area (VTA), dopaminergic (DA) neurons that project to the nucleus accumbens (NAcc) and are involved in reward processes, LEAP2 attenuates the release of dopamine in the NAcc and reduces reward-related behaviors. In situations of negative energy balance, the levels of LEAP2 are reduced whereas in situations of positive energy balance, the levels of LEAP2 are elevated. LEAP2 levels are also elevated in pathological conditions associated with chronic undernutrition such as Anorexia Nervosa and Amyotrophic Lateral Sclerosis and by chronic inflammation. LEAP2 is also finely tuned by the nutrients type and is elevated in the post-prandial state.</figcaption></figure>
hindbrain ghrelin-stimulated feeding being blocked by LEAP2 [27,34]. In the ventral hippocampus, ghrelin can reduce the effects of gut-derived satiation signals to increase meal size through a downstream orexin signalling pathway to the hindbrain laterodorsal tegmental nucleus, and can initiate eating by GHSR mediated increase in post-synaptic inhibition of ventral hippocampal neurons projecting to the nucleus accumbens (NAcc) [35,36]. It also modulates cognitive aspects of feeding by linking memory-related hippocampal circuits to orexin-producing neurons in the lateral hypothalamus [37–39]. Ghrelin also modulates feeding behaviour and food choice in the lateral parabrachial nucleus (IPBN) possibly by interfering with glutamatergic neurons expressing the GHSR [40]. Feeding regulation is also influenced by activation of IPBN astrocytes, which are involved in modulating the effects of ghrelin [41]. Exploring the role of LEAP2 in these different cerebral sites appears important given the multiple targets of ghrelin action.

## 2.3. Role of LEAP2 in the regulation of growth hormone secretion and response to starvation

Besides stimulating food intake, one of the best characterized functions of ghrelin is its ability to induce GH secretion by binding to GHSR [11], acting directly on the pituitary gland to release GH but also indirectly by stimulating hypothalamic GHRH neurons excitability and GHRH release [42] and by inhibiting somatotropin release-inhibiting hormone (SRIH) release [43]. In mice, ip administration of LEAP2 abolishes ghrelin-induced GH release from the pituitary gland, while treatment with LEAP2 neutralizing antibodies increases the amount of GH released following a 24-hour fast [6]. Ghrelin mediated stimulation of GH secretion has been described as fundamental to maintain euglycemia in conditions of starvation, as GH is able to stimulate glycogenolysis and gluconeogenesis [44,45], although this action of GH has been debated [46,47]. Furthermore, ghrelin is known to have a hyperglycaemic effect, even under homeostatic conditions [48]. Accordingly, LEAP2 administration significantly reduced blood glucose in mice after an *ad libitum* meal, an effect that was not observed in *Ghsr* KO mice [49]. Furthermore, mice exposed to a 40 % reduction of their *ad libitum* caloric intake exhibit critically low blood glucose levels when exposed to lasting systemic over-expression of LEAP2 mediated by an adeno-associated viral system, and this is partially rescued by concomitant delivery of GH through osmotic pumps [6]. Consistently, chronic sc LEAP2 delivery via osmotic pump in mice exposed to a 40 % caloric restriction of their *ad libitum* intake resulted in a significant decrease in blood glucose compared to vehicle-treated mice, associated with a significant decrease in body weight after one week of treatment [17]. Similar observations have been made in humans: LEAP2 infusion in healthy men fed with an *ad libitum* meal induced significantly lower postprandial GH levels and blood glucose peaks compared to saline infusion. In addition, LEAP2 infusion increased preprandial levels of insulin and C-peptide, and of the C-peptide/glucose ratio, suggesting an insulinotropic effect of the hormone [49], which is consistent with the well characterized decrease in insulin levels induced by ghrelin [48].

# 3. Regulation of LEAP2: role of energy status, nutrients and beyond

## 3.1. Long-term regulation of LEAP2 by positive and negative energy balance

The regulation of LEAP2 by positive and energy balance has been characterized in both rodents and humans [19] (Fig. 1). Indeed, plasma LEAP2 was found to be elevated in severely obese humans and obese mice, while ghrelin was regulated in the opposite way. Furthermore, plasma LEAP2 concentrations were found to be positively correlated with body mass index, body fat accumulation, fasting glucose and triglycerides. Since, several other studies have shown that plasma LEAP2 concentrations were associated with overweight and obesity [26,48].

Plasma LEAP2 concentration correlates with glycaemia in normal weight subjects, whereas in overweight/obese subjects, it is associated with body mass index, waist circumference and obesity, while plasma ghrelin concentration negatively correlates with body mass index (BMI) [28]. Interestingly, LEAP2 expression is increased by high cholesterol in a human hepatoma cell line [50], suggesting that increased LEAP2 in overweight and obese adults may be mediated in part by elevated cholesterol. In contrast to studies in adult obesity correlating plasma LEAP2 concentrations with body mass index, a study in obese children aged 3–12 years old reported lower plasma LEAP2 concentrations compared to normal weight controls [51]. This suggests that, while higher LEAP2 in obese adults may be a compensatory mechanism for excessive weight gain, lower LEAP2 levels in obese children could be the cause of overeating, although the relationship remains to be established.

Opposite adaptations of LEAP2 plasma levels are observed in conditions of negative energy balance. Plasma LEAP2 concentrations are reduced by fasting, long-term calorie restriction and weight loss in rodents [14,49]. In chow-diet fed male and female mice, vertical sleeve gastrectomy (VSG) decreases body weight, blood glucose and plasma ghrelin concentrations while increasing plasma LEAP2 concentrations and hepatic LEAP2 mRNA expression. In contrast, in mice fed with a high fat diet (HFD), VSG is associated with decreased plasma LEAP2 concentrations [52]. Similar results emerge from clinical studies in patients with obesity. LEAP2 plasma levels decreased with weight loss induced by Roux-en-Y gastric bypass in obese subjects [19,53–55]. The regulation of LEAP2 after long-term dietary weight loss has also been explored after 2 years of follow-up after either standard medical management (SMM, 600 kcal/day deficit) or duodenal-jejunal bypass liner insertion (DJBL) in adults with obesity and type 2 diabetes. In SMM, fasting plasma LEAP2 was decreased from baseline and was positively correlated with BMI, glycated hemoglobin A1c, fasting plasma glucose, serum insulin, homeostasis model assessment (HOMA) index and serum triglycerides [56]. In DJBL, the amplitude of decrease of plasma LEAP2 was lower and delayed compared to SMM, which may explain greater weight loss.

## 3.2. Short-term regulation by meals, nutrients and dietary status

Ghrelin secretion is finely tuned by the timing of meals, plasma ghrelin concentrations being elevated in anticipation of meals in humans [57,58], which has been associated with increased hunger in humans [58], as well as in rodents [59,60], while they are decreased postprandially. Postprandial ghrelin secretion is differentially regulated by the type of diet; the postprandial decrease in plasma ghrelin concentrations is greater with carbohydrates than with lipids and greater with lipids than with proteins [61]. LEAP2 appears to be regulated in an opposite manner to ghrelin in situations of positive or negative energy balance, and is also regulated in the course of a meal and by the nature of the diet/nutrients, although the amplitude of variations appears to be much smaller than that of ghrelin. In healthy humans, plasma LEAP2 increases by 20 to 50 % during the postprandial period [29], which correlates positively with decreased appetite and food cues responsiveness. This postprandial increase in LEAP2 was also observed in obese subjects 12 to 18 months following vertical sleeve gastrectomy to induce weight loss [19]. This suggests that LEAP2 may be involved in satiation. Consistent with this hypothesis, a reduction in food intake was observed after LEAP2 administration, which achieves mildly supraphysiological plasma LEAP2 concentrations during an *ad libitum* test lunch in men, although appetite ratings were unchanged [49]. The relationship of LEAP2 with hunger during a test meal has also been investigated in obese subjects using visual analogue scales. Preprandial plasma LEAP2 concentrations were 1.6-fold higher while postprandial plasma LEAP2 concentrations increased by 58 % in overweight/obese females. Importantly, preprandial plasma LEAP2 concentrations were inversely correlated with hunger scores [28]. The time course of postprandial LEAP2 concentrations in response to a standardized meal was
investigated in diabetic and non-diabetic subjects. While plasma LEAP2 concentrations increase at 1- and 2-hours following meal consumption, plasma ghrelin concentrations decrease only after 1 hour. Interestingly, the post-meal increase in plasma LEAP2 concentrations is delayed and occurs at 2 h following meal consumption in diabetic subjects compared to subjects with normal glucose tolerance [52]. Finally, plasma LEAP2 has been studied in chronic diseases associated with loss of appetite and weight loss such as amyotrophic lateral sclerosis (ALS). Postprandial ghrelin levels were reduced by 52 % in cases compared to controls and the LEAP2/ghrelin molar ratio was increased by 249 %, which may indicate ghrelin resistance in this condition. Patients with lower LEAP2/ghrelin ratios tended to have better functional capacity at assessment. This suggests a loss of ghrelin action in patients with ALS, which may contribute to an impaired ability to tolerate the physiological effects of the disease [62]. Similarly, LEAP2 has been implicated in cancer-related anorexia cachexia syndrome (CACS): LEAP2 deletion in mouse models of the disease ameliorates the phenotype by increasing food intake and decreasing weight loss [63]. In line with these findings, in cancer patients, higher LEAP2/ghrelin ratios are correlated with the degree of weight loss in the six previous months, possibly suggesting a detrimental role of LEAP2 in CACS [63]. Finally, as LEAP2 production appears to be positively regulated by both acute and chronic inflammation [5,64], it would be interesting to further explore whether this influences the reduction in appetite associated with these diseases.

The regulation of LEAP2 during the course of a meal has also been studied in rodents [65]. Comparing acute dietary challenges using various nutrient sources (mixed meal, glucose, olive, lard and fish oil), jejunal LEAP2 expression was increased by all dietary challenges, except fish oil, whereas liver LEAP2 expression was increased only following the mixed meal. Plasma and portal vein LEAP2 concentrations, were also increased by meal challenges, but the smallest increase was observed following fish oil [65]. The effect of specific fatty acids (oleic, docosahexaenoic, and linoleic acid) was also tested in mouse intestinal organoids. Oleic acids but not docosahexaenoic acids increased LEAP2 expression [66]. Interestingly, LEAP2 expression in a human hepatoma cell line was found to be reduced by treatment with polyunsaturated fatty acids treatment [50], suggesting a possible role of triglycerides in reducing postprandial plasma LEAP2 concentrations. Furthermore, hepatic LEAP2 expression correlates with hepatic glycogen levels, suggesting that high carbohydrate-induced accumulation of liver glycogen may be involved in increasing LEAP2 expression. In contrast, plasma LEAP2 concentrations decrease after a high-protein (35 % of energy) meal in humans, where the protein content is around 12 to 18 % in the previous studies discussed above. Consistent with those observations, a recent study assessing the mouse hepatic transcriptome found that LEAP2 expression decreased with high-protein meals (>20 %) [67].

Oral administration of corn oil or palmitate also increased plasma LEAP2 levels and hepatic LEAP2 mRNA levels [52]. Palmitate and glucose increased LEAP2 mRNA expression in primary mouse hepatocytes, suggesting that they directly regulate LEAP2 expression in the liver. In intestinal organoids, oleic acid increased LEAP2 mRNA expression, but linoleic acid did not [65]. Corn oil is rich in oleic acid (30 % of total calories) and palmitate (10 % of total calories), which may contribute to increased LEAP2 expression in the liver.

Finally, chronic modifications in dietary status such as long-term consumption of a HFD also influence baseline and meal-associated LEAP2 levels. Indeed, HFD increased plasma and liver LEAP2 mRNA expression and decreased plasma ghrelin, together with the development of glucose intolerance, which was normalized when mice were switched to a standard chow diet [60,61]. Furthermore, elevation of plasma LEAP2 following olive oil but not fish oil meal challenge was greater in HFD fed mice than in chow-fed mice [65].

### 3.3. Regulation by glucose status and substrates of glucose metabolism

Consistent with LEAP2 being down-regulated by states of energy

deprivation, plasma LEAP2 concentrations are reduced following short-term or prolonged fasting in mice [7,19], an effect that is mediated in part by reduced liver LEAP2 expression, or after exposure to a ketogenic diet for 3 weeks in mice. Interestingly, increased beta-hydroxybutyrate (BHB), an intermediate product of fatty acids oxidation and a marker of ketosis during states of starvation may be a mechanism by which fasting reduces plasma LEAP2. Indeed oral administrations of BHB in rodents is associated with reduced LEAP2 expression in the liver and intestine, as well as reduced plasma levels of LEAP2 [54]. Similarly, in healthy men, plasma LEAP2 concentrations are reduced following a prolonged 36 h fast or endurance exercise, conditions associated with increased plasma BHB, and by both oral and intravenous BHB administration [54,68].

While plasma LEAP2 concentrations and hepatic LEAP2 mRNA are elevated by oral glucose or lactate administration, they are reduced by intravenous glucose or lactate in mice [68]. High glucose concentrations have been shown to increase LEAP2 concentrations in the hepatic vein and abdominal aorta [52]. However, in humans, a positive correlation between LEAP2 and glucose levels is only observed in obese individuals, both after a fast and post-prandially [19]. On the contrary, in healthy individuals there is no clear evidence that increased LEAP2 secretion during the postprandial period is regulated by glucose, insulin or triglycerides because LEAP2 does not correlate with any of these factors [29].

## 4. Clinical implications in metabolic and eating disorders: from pathophysiology to therapeutic perspectives

### 4.1. LEAP2 variants that may affect LEAP2 levels and feeding behavior

The N-terminal portion of LEAP2 is essential for both receptor binding and microbial membrane affinity [69,3]. By assessing the receptor binding affinity and biological activity of engineered LEAP2 fragments of different lengths, M'Kadmi and colleagues demonstrated that the binding determinant of LEAP2 resides at the 1-8 N-terminal sequence, but that a longer segment containing at least the 1-12 N-terminal residues had increased binding potency and efficacy [18]. A subsequent alanine-scanning mutagenesis study confirmed that the key residues for LEAP2 binding are Thr2, Phe4, Trp5, and Arg6. In contrast, the C-terminal end appears to be irrelevant for both antibacterial activity and GHSR binding, but may play a role in stabilizing LEAP2 in the plasma through the formation of two disulfide bonds (Cys54-Cys65 and Cys60-Cys70) [70].

LEAP2 is a gene located on chromosome 5 (5q31.1). The probability of being loss-of-function intolerant (pLI)<fnref n="1" /> [71] of this gene is 0 suggesting that it is highly tolerant to variants. In fact, 26 different rare loss-of-function variants (non-sense; frameshift; splice) have been reported in the gnomADv4.1 database, a resource aggregating exome and genome sequencing data from a wide variety of large-scale sequencing projects (https://gnomad.broadinstitute.org). The most common is a frameshift variant called p.(Pro26GlnfsTer), which has been identified on 33 alleles out of 1,514,100 alleles from «controls». 29 of these 33 alleles are from African/African-American individuals. However, no homozygotes were identified in the «controls». In addition, 108 different missense variants, 2 start-lost and 2 stop-lost variants were identified in the human database gnomADv4.1. Variants were identified in the different domains of the protein: signal (amino acids 1-22), propeptide

<footnote n="1">¹ The "probability of loss-of-function intolerance" score (pLI) has been established to estimate the probability that loss of function (LoF) in one gene allele causes a haploinsufficient phenotype and estimates the likelihood that a gene is intolerant to a variant that produces LoF in the protein product. The score is derived using the gnomAD database that is an extensive catalogue of human genetic diversity. pLI was previously shown to separate genes of adequate length into those intolerant (pLI ≥ 0.9) or tolerant (pLI ≤ 0.1) to LoF.</footnote>
(amino acids 23–37), chain (amino acids 38–77). The disulfide bonds involve amino acids 54, 65, 60 and 70. The chain starts at position 38. The amino acid sequence of the mature protein is: MTPFWRGVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE.

Interestingly, some missense variants were identified in key positions of the mature protein and thus may possibly affect the structure and the function of the LEAP2 peptide. Two specific coding variants appear to be relatively common: p.(Met11Val) (1180 over 1,614,052 alleles, including 1077 alleles positive from individuals from East Asian individuals) and p.(Gly22Asp) (4072 over 1,624,064 alleles, including 2978 alleles positive from African/African American individuals) (http s://gnomad.broadinstitute.org). Some genetic non-coding variants such as rs765760 and rs803223 have been reported to influence plasma LEAP2 levels [51,72]. Furthermore, the rs57880964 G allele was associated with higher BMI and BMI-adjusted waist-to-hip ratio in the total population and in women [72,73]. The interaction between LEAP2 and GHSR involves at least the Phe4 and Trp5 of the mature LEAP2 (aa 38–77) [70]. Different rare missense variants at these positions have been described in the general population: p.(Phe41Leu) (14 over 1,613, 758 alleles), and p.(Phe41Val) (2 over 1,613,876 alleles) (http s://gnomad.broadinstitute.org). The amino acid substitution leads to a loss of the hydrogen bond between LEAP2 and GHSR, suggesting that these rare missense variants may affect the antagonistic effect of LEAP2 on its receptor. Since the loss-of-function variants of LEAP2 reduces its ability to interact with GHSR, this could be associated with an enhanced effect of ghrelin, whereas the gain-of-function variants may reduce the effect of ghrelin, which would deserve further investigations.

## 4.2. LEAP2 as a new therapeutic target in obesity

The effects of LEAP2 on feeding sparked interest in the molecule as a potential target for the treatment of obesity, leading to several studies focusing on the role of LEAP2 in both genetic and environmental mouse models of the disease. Obesity can be induced in mice by feeding them a high-calorie diet containing at least 40 % of fat (HFD). These mice will develop diet-induced obesity (DIO), which is characterized by increased body weight, adipocyte hyperplasia and hypertrophy due to increased lipogenesis, low-grade inflammation, increased blood glucose and insulin levels, eventually leading to insulin resistance, increased serum triglycerides and cholesterol concentrations, and hepatic steatosis due to intracytoplasmic accumulation of triglycerides [74].

Acute icv LEAP2 treatment in DIO mice fed a HFD (60 % fat) for 12 weeks has been shown to reduce food intake and blood glucose levels during refeeding after a fast [25]. Similarly, adeno-associated viral mediated overexpression of LEAP2 in the hypothalamic ArcN significantly lowered body weight, food intake and fat mass in DIO mice compared to controls, which was associated with a reduction in the size of epidydimal white adipose tissue, a decrease in blood cholesterol levels and in hepatosteatosis, suggesting improved liver health [26]. Sc injections of palmitoylated LEAP2 (1–14) twice a day for two weeks in DIO mice that had been exposed to HFD for 4 months were shown to improve liver health as well by attenuation of de novo lipogenesis, even though no effects on food intake, cholesterol and triglyceride levels were observed, suggesting beneficial effects on metabolism beyond weight loss and food intake modulation [75]. Consistently, Leap2 KO female mice fed with a HFD (42 % fat Western Diet) for 16 weeks showed increased body weight and a higher number of large lipid droplets in the liver [21]. The hepatoprotective effect of LEAP2 has been further highlighted in wild-type and Leap2 KO mice fed the Gubra Amylin NASH (GAN) diet, i. e., a high fat (40 %) and high fructose (20 %) diet that leads to obesity and metabolically associated fatty liver disease (MAFLD) in mice. While Leap2 KO mice showed a higher prevalence of more severe steatosis condition, daily injections of a long-lasting LEAP2 analogue, not only reduced body weight, but also eliminated more severe grades of fibrosis than the milder grades, and this was accompanied by reductions in hepatic triglycerides, plasma total cholesterol, alanine transaminase

(ALT), microvascular steatosis, lobular inflammation and non-alcoholic steatohepatitis (NASH) activity score [76]. Similar improvements were also observed in wild-type mice treated with the LEAP2 analogue [76].

LEAP2 also affects the liver in mice fed a standard diet: chronic peripheral LEAP2 infusion using osmotic pumps in mice food restricted to 40 % of their *ad libitum* food intake resulted in a significant increase in the expression of the pro-inflammatory cytokines interleukin 6 (IL-6) and interleukin 1β (IL-1β), and this was accompanied by a significant decrease in body and liver weight. This suggests an inflammatory effect of LEAP2 in the liver during caloric restriction [17]. The pro-inflammatory effects of LEAP2 in conditions of energy deficit strikingly differs from the beneficial effect of LEAP2 on inflammation in MAFLD. Different mechanism may be involved since the pro-inflammatory effect of LEAP2 during calorie restriction is also observed in Ghsr KO mice indicating that this effect is GHSR-independent [17], while LEAP2-induced reduced inflammation in MAFLD may be the consequence of reduced body weight and improved metabolic state.

## 4.3. LEAP2 in hedonic and rewarding components of food intake and its role as a potential biomarker in eating disorders

Beyond being implicated in homeostatic feeding, LEAP2 has been involved in hedonic and rewarding aspects of eating behaviour. A key neurobiological substrate in this context is the ventral tegmental area (VTA), a region that contains dopaminergic neurons expressing GHSR and that acts as an input for the mesocortical reward circuit. Ghrelin-mediated activation of GHSR in this neuronal population leads to DA release in the nucleus accumbens (NAcc), which is associated with rewarding food-seeking behaviour [77,78]. Interestingly, icv injection of LEAP2 in mice leads to a significant decrease in the consumption of palatable food such as peanut butter, Nutella® and chocolate, and reduced preference for peanut butter as compared to chow. In addition, LEAP2 decreased memory conditioned place preference for palatable food suggesting impairments in reward-related memory retrieval associated with palatability, and this was accompanied by a significant decrease in palatable-food elicited dopamine release in the NAcc [79]. Consistent with a role of LEAP2 in reducing hedonic feeding, icv LEAP2 injection decreased consumption of HFD in a mouse model of binge-eating in which access to highly palatable food was restricted to two hours per day [80]. Ghrelin-dependent activation of the VTA and subsequent dopamine release in the NAcc is also involved in reward processes beyond feeding, including alcohol consumption. In particular, ghrelin infusion into the VTA has been shown to increase alcohol consumption in rodents, while GHSR inhibitors decrease it [81]. Consistently, icv delivery of LEAP2 in mice attenuates the memory of conditioned place preference for alcohol, and this is again associated with a suppression of dopamine release in the NAcc. In addition, central icv LEAP2 administration reduces alcohol intake in male rats that have been exposed to the substance for the previous six weeks [82].

These findings on the ability of LEAP2 to decrease the salience of rewarding stimuli in rodents are consistent with human data. In overweight and obese individuals, LEAP2 concentrations negatively correlate with reward-responsiveness elements of the behavioural inhibition system and behavioural activation system (BIS/BAS) scale, suggesting that the peptide blunts responsiveness to rewarding stimuli such as palatable food [28].

Based on the ability of LEAP2 to antagonize food intake and hedonic feeding and the influence of nutritional status on plasma LEAP2 concentrations, it can also be postulated that altered LEAP2 regulation may influence feeding behavior in pathological conditions and thus the susceptibility to eating disorders. Eating disorders involve not only homeostatic alterations but also dysfunctions in food reward and decision-making processes, on which the role of LEAP2 is still largely unknown. Interestingly, one study explored LEAP2 inhibition of ghrelin-induced feeding in rats exposed to time-restricted feeding. Central icv infusion
of LEAP2 successfully decreased ghrelin-induced food intake in freely-
fed animals, this effect being blunted when animals have been
exposed to 28 days of time-restricted feeding with access to food only
during the light phase [24]. These results suggest that adequate LEAP2
regulation can be impacted by disordered eating. Another study
explored the behavioural response to ghrelin and LEAP2 infusion
measured with cumulative food intake in mice after 2 and 8 weeks on a
HFD [66]. Two weeks on HFD was sufficient to blunt ghrelin-induced
food intake compared to mice exposed to standard chow diet. After 8
weeks, HFD fed mice did not exhibit changes in food intake after both
ghrelin or LEAP2 injection compared to saline injection which was
reversible after 2 weeks of standard chow in the previously HFD-fed
animals. These results suggest that modification of eating habits can
induce disrupted adaptive mechanisms between metabolic sensing and
food intake, possibly participating to the persistence of abnormal eating
behaviour.

Among eating disorders, AN is characterized by chronic food re-
striction and extremely low body weight leading to dramatic health
consequences, both somatic and psychological [83]. It has been pro-
posed that AN is a metabopsychiatric disorder, meaning that AN has a
psychiatric as well as a metabolic etiology [84]. Plasma ghrelin con-
centration are chronically elevated in patients with AN as a response to
starvation [85,86]. However, patients do not show the normal physio-
logical response to this increased orexigenic signal which would be to
increase appetite. Consistently, exogenous ghrelin administration does
not increase food intake in these patients [87,88]. While this is consis-
tent with defective GHSR signaling, other relevant behaviors typical of
ghrelin action appear to remain intact, such as heightened anxiety and
motivation for (and preoccupation with) food. Interestingly, in a lon-
gitudinal study of patients with restrictive-type AN, LEAP2 plasma
concentrations were found to be higher in the undernourished than in
the refed state, so the possibility that chronically elevated LEAP2 may be
involved in restrained eating or other symptoms of AN, by counteracting
the actions of endogenous ghrelin, should be further investigated [53].
Interestingly, after 6-months of follow-up, only patients with unstable
weight gain, but not those with stable weight gain, had significantly
lower plasma LEAP2 concentrations after the inpatient refeeding pro-
gram, suggesting that LEAP2 may also predict relapse.

The dopamine reward system, which is targeted by ghrelin [81,89],
has been previously explored or discussed in the context of anorexia
nervosa [90-96]. Whether LEAP2 might also be associated with specific
behaviors of AN patients has not been well studied so far. LEAP2 seems
to predict gambling disorders, which are characterized by impaired
decision making, low cognitive flexibility and high impulsivity. In
controls, high LEAP2 plasma levels are associated with poor cognitive
performance and low plasma ghrelin levels with reduced cognitive
flexibility and perseverative errors on the Wisconsin card sorting test
(WCST) and Iowa gambling task (IGT) [97].

Altogether, these data suggest a role of LEAP2 in processes that
regulate reward and implicates LEAP2 in eating disorders. However,
there are still some debates on how peripheral ghrelin and LEAP2 may
access and bind GHSR located in deep brain regions such as the VTA,
thus the exact pathways by which LEAP2 regulates reward structures
requires further investigations.

**5. Conclusion**

The ghrelin signaling system in the brain is strongly orexigenic,
affecting both appetitive and consummatory behaviors as well as
metabolic responses to efficiently adapt our needs to changes in energy
status. LEAP2 is now emerging as an opposing signal to ghrelin's action
on neural circuits involved in hunger processes, exerting its effects via
the GHSR. One important target sites of action of LEAP2 are the NPY/
AgRP and POMC neurons in the hypothalamic arcuate nucleus, a key
regulator of homeostatic feeding. Beyond its role in homeostatic feeding
and metabolic responses, LEAP2 also appears to play a role in the

regulation of circuits regulating food reward processes and may be
involved in eating disorders, although the exact mechanism needs to be
further investigated. Importantly, while LEAP2 was initially character-
ized as an antagonist at the GHSR, it may also interact with the GHSR by
modulating its constitutive activity and/or heterodimerize with other
relevant G-protein receptors, such as the dopamine DRD2. Better un-
derstanding how LEAP2 modulates the GHSR and on which exact cir-
cuits is essential to consider new therapeutic targets for metabolic and
eating disorders.

Web references

https://gnomad.broadinstitute.org, 07/03/2025

CRediT authorship contribution statement

Francesca Mariuz: Writing – review & editing, Writing – original draft. Tatiana Landré: Writing – original draft. Chloé Tezenas du Montcel: Writing – review & editing. Alexandre Benani: Writing – review & editing. Thierry Bienvenu: Writing – review & editing. Virginie Tolle: Writing – review & editing, Supervision.

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