Ghrelin–LEAP2 interactions along the stomach–liver axis

Abstract

Ghrelin produced in the stomach promotes food intake and GH secretion, and acts as an anabolic peptide during starvation. Ghrelin binds to the growth hormone secretagogue receptor, a G protein-coupled receptor (GPCR), whose high-resolution complex structures have been determined in the apo state and when bound to an antagonist. Anamorelin, a low-molecular-weight ghrelin agonist, has been launched in Japan for the treatment of cancer cachexia, and its therapeutic potential has attracted attention due to the various biological activities of ghrelin. In 2019, liver-expressed antimicrobial peptide (LEAP2), initially discovered as an antimicrobial peptide produced in the liver, was identified to be upregulated in the stomach of diet-induced obese mice after vertical sleeve gastrectomy. LEAP2 binds to the GHSR and antagonizes ghrelin’s activities. The serum concentrations of human LEAP2 are positively correlated with body mass index, body fat accumulation, and fasting serum concentrations of glucose and triglyceride. Serum LEAP2 elevated and ghrelin reduced in obesity. Ghrelin and LEAP2 regulate body weight, food intake, and GH and blood glucose concentrations, and other physiological phenomena through their interactions with the same receptor, GHSR.

Introduction

The year 2024 marks the twenty-fifth anniversary of the discovery of ghrelin, an orexigenic peptide secreted exclusively from the stomach. Research has been conducted for years regarding the potential existence of a peptide modulating ghrelin’s actions. In 2019, liver-expressed antimicrobial peptide 2 (LEAP2) produced in the liver was shown to counteract the activities of ghrelin via its receptor, the growth hormone secretagogue receptor (GHSR). Animal experiments and human clinical studies have demonstrated the functional interactions between ghrelin and LEAP2. Here we review recent topics in ghrelin research, including the discovery of LEAP2, and also LEAP2–ghrelin interactions along the stomach–liver axis.

1. Ghrelin

1-1. The production of ghrelin

In 1999, Kojima and Kangawa discovered an endogenous ligand peptide for GHSR and named it “ghrelin” [1]. Human ghrelin contains 28 amino acids, among which the third serine residue is acylated by the addition of a medium-chain fatty acid, octanoic acid [1] (Fig. 1a). The protein resulting from the absence of this acylation is named desacyl ghrelin. Ghrelin is primarily secreted from X/A-like cells, which are mostly found in the gastric body of the stomach [2]. These cells were discovered in the 1950s, and while they were shown to contain abundant storage granules characteristic of endocrine cells, the nature of the stored substances was unknown. Ghrelin O-acyltransferase (GOAT), a member of membrane-bound O-acyltransferase family, catalyzes the transfer of octanoic acid to the third serine of ghrelin [3] (Fig. 1). GOAT utilizes medium-chain fatty acids contained in the diet as substrates [4]. The ratio of ghrelin to desacyl ghrelin in human blood is nearly 1:10 (ghrelin: 10–20 fmol/mL, desacyl ghrelin: 100–150 fmol/mL) [5]. Desacyl ghrelin also exhibits biological activities that are not mediated via GHSR, and its cognate receptor has yet to be identified [6-9].

Fig. 1  Amino acid sequences of human ghrelin and LEAP2

(a) Ghrelin is produced by the addition of octanoic acid to serine at position 3. (b) LEAP2 has two disulfide bonds and its 10-amino acid N-terminal region is a biologically active site.

Ghrelin has a variety of biological activities, including those related to feeding and body temperature regulation [10, 11]. Ghrelin is more potent than growth hormone-releasing hormone in terms of GH release [12]. Ghrelin is the only peptide that transmits peripheral hunger signals to the brain [13]. Ghrelin exhibits potent hyperphagic effects by activating orexigenic neurons, specifically neuropeptide Y (NPY) neurons and agouti-related protein (AgRP) neurons in the hypothalamic arcuate nucleus [10]. Ghrelin inhibits the release of α-melanocyte stimulating hormone (α-MSH), an anorectic neuropeptide cleaved from proopiomelanocortin (POMC) [14]. Leptin secreted by adipocytes reduces feeding by suppressing NPY and AgRP neurons and activating POMC neurons [15]. Ghrelin and leptin regulate feeding by affecting these neurons in opposing ways [10]. Ghrelin production is upregulated under nutrient-deficient conditions such as starvation, cachexia, anorexia nervosa, and cancer [11, 16]. By contrast, its production is downregulated by gastrectomy and by some gastric disorders that cause the loss of ghrelin-producing cells [17-19]. This has led to the development of a low-molecular-weight ghrelin agonist, anamorelin, as a drug for cancer cachexia as described below.

Ghrelin is also involved in the regulation of body temperature and sleep [20, 21]. Wild-type mice under food restriction were found to enter torpor, a hibernation-like state in which body temperature is temporarily reduced [22], whereas ghrelin knockout (Ghrl^–/–) mice did not exhibit reduced body temperature or enter torpor [22]. Desacyl ghrelin also lowered body temperature [23, 24].

1-2. Ghrelin regulates feeding behavior through the vagus nerve

Gut hormone signaling via the vagus afferent nerve is a critical pathway by which the gastrointestinal tract interacts with the brain to regulate energy balance and metabolic functions [25-28]. The nodose ganglion (NG) is a constellation of primary sensory neurons that serve as vagal afferents. Vagal sensory neurons are molecularly heterogeneous and exert diverse physiological functions, including feeding regulation [29-31]. NG neurons relay gastrointestinal tract-derived signals to the nucleus tractus solitarius in the medulla oblongata, where secondary neurons relay the information to particular regions of the brain [11, 32]. NG neurons have been shown to innervate the gastrointestinal tract and pancreas [33, 34]. Specifically, the right and left NGs innervate the ventral and dorsal portions of the stomach, respectively [33, 34]. In mice, nearly equal numbers of left and right NG neurons receive stomach-derived signals [35]. Left NG neurons convey sensory information from the liver [33, 36]. Several lines of evidence have elucidated the neurobiological mechanisms whereby vagal afferent neuron signaling regulates feeding. Some vagal afferent terminals are located close to ghrelin-producing cells in the mucosal layer of the gastric body [13, 37]. Direct electrophysiological measurements showed that ghrelin attenuated vagal afferent activity [37-40]. By contrast, the anorectic peptides glucagon-like peptide 1 (GLP-1) and cholecystokinin enhanced vagal afferent activity [35, 41].

Individual NG neurons express multiple receptors that contribute to the regulation of feeding [42]. GHSR synthesized in the NGs is transported to the afferent terminals innervating the gastrointestinal tract [35, 43-45]. We demonstrated that 70.8% of left and right NG neurons in mice expressed both GHSR and GLP-1R [35]. Patch-clamp experiments combined with single-cell polymerase chain reaction analysis in isolated NG neurons showed that ghrelin generated a hyperpolarizing current while GLP-1 generated a depolarizing current [35]. Ghrelin and GLP-1 interact in the regulation of feeding through their opposing effects on the cellular excitability of vagal afferent neurons [35]. Ghrelin-induced food intake in rats was abolished by administering a K_ATP channel antagonist or by silencing the Kir6.2 channel subunit, a critical component of a potassium channel in the NG [24]. Ghrelin administration to mice induced extracellular signal-regulated kinases 2 (Erk 2) phosphorylation in the NG [45]. The administration of Gαi inhibitor pertussis toxin to the NG, or silencing of phosphatidylinositol 3-kinase (PI3K) or Erk1/2, abolished ghrelin-induced hyperpolarization in Chinese hamster ovary cells [45, 46]. Collectively, ghrelin activates the Gαi-PI3K-Erk1/2-K_ATP pathway, evokes potassium currents, and causes hyperpolarization, thereby suppressing vagal afferent activity (Fig. 2).

Fig. 2  Schematic illustration of the vagus afferent nerve conveying gastric-derived ghrelin signals

Vagal afferent neurons are located in the bilateral NG neurons. Ghrelin activates the Gαi-PI3K-Erk1/2-K_ATP pathway in NG neurons, and evokes potassium currents, thereby causing hyperpolarization. By contrast, GLP-1 induces depolarization. Ghrelin and GLP-1 regulate feeding through their opposing effects on the cellular excitability of vagal afferent neurons. ARC, arcuate nucleus; NG, nodose ganglion; NTS, nucleus tractus solitarius.

1-3. Structure of GHSR

Recent technological innovations in structural biology have revealed both active and inactive forms of GHSR. The structure of antagonist-bound GHSR was determined by X-ray crystallography in 2020 [47]. GHSR has a typical structure, consisting of a G protein-coupled receptor (GPCR) with seven transmembrane helices (TM1–7) and a short intracellular amphipathic helix 8 (Fig. 3a). The pocket of GHSR has a characteristic structural feature that is not found in any GPCRs: a bifurcated pocket is divided in two by the formation of a salt bridge between the Glu124^3.33 of TM3 and the Arg283^6.55 of TM6 (superscript denotes Ballesteros–Weinstein numbering). GHSR antagonists occupy both pockets across the salt bridge (Fig. 3b). Structural analysis of ghrelin-bound GHSR has demonstrated that the ghrelin peptide chain coordinates to cavity I, while octanoic acid coordinates to cavity II [48-50] (Fig. 3c).

Fig. 3  (a) The overall structure of antagonist (compound 21)-bound GHSR (PDBID: 6KO5). GHSR and the antagonist are shown in cyan and yellow, respectively. (b) The ligand-binding pocket of GHSR. The antagonist straddles the salt bridges formed by Glu124^3.33 and Arg283^6.55, and fills the two cavities. (c) The active ghrelin-binding mode (PDBID: 7F9Y). The peptide chain of active ghrelin and octanoic acid are shown in yellow and red, respectively. The peptide chain coordinates to cavity I (blue dashed line) and octanoic acid coordinates to cavity II (green dashed line).

The following fundamental mechanism of GPCR activation has been proposed. When agonists bind to GPCR, they promote the rearrangement of Trp276^6.48 [51]. This movement propagates downwards in TM6, and the intracellular region of TM6 spreads outwards. Consequently, the intracellular region of GPCR adopts a more open conformation, allowing access for G proteins and thereby activating intracellular signaling pathways. In the case of GHSR, neither ghrelin nor small-molecule agonists directly interact with the receptor’s Trp276^6.48. Currently, the activation mechanism of GHSR is most commonly thought to consist of the following [48]: Arg283^6.55 of GHSR, which forms a salt bridge, is pushed downward by the agonist, and this leads to the reorientation of aromatic amino acids (Phe279^6.51, His280^6.52, and Phe312^7.42). This rearrangement of the aromatic cluster reorients Trp276^6.48, resulting in an outward shift of TM6 (Fig. 4a). Therefore, Trp276^6.48 in GHSR is indirectly reoriented by the structural changes resulting from the salt bridge. The salt bridge in the ligand-binding pocket of GHSR could play a pivotal role in both GHSR activation and in the formation of the bifurcated pocket that is engaged in ligand recognition. Most ghrelin receptor agonists and antagonists are coordinated to straddle the salt bridge in the ligand-binding pocket. However, GHSR agonists, in particular, stabilize the GHSR in its active conformation by inducing a downward movement in the salt bridge.

Fig. 4  (a) The activation mechanism of GHSR. GHSRs bound to an inverse agonist (PF-05190457), an antagonist (compound 21), and an agonist (ghrelin) are shown in green, cyan, and magenta, respectively. The intracellular region of TM6 opens outward by an interlocking motion initiated by a downward shift of Arg283^6.55 that results in the formation of a salt bridge. (b) The pharmacological definition of GPCR. Full agonist, partial agonist, neutral antagonist, and inverse agonist are shown in red, orange, cyan, and green, respectively. Antagonists suppress receptor activation by agonists. Inverse agonists reduce receptor activity below that in the basal state.

Structural analysis of inverse agonist-bound GHSR has provided additional insights. Some GPCRs transmit signals into the cell even in the absence of agonist binding, which is referred to as constitutive activity. Inverse agonists reduce receptor activity below that in the basal state (Fig. 4b). Among GPCRs, GHSR has particularly high constitutive activity, exhibiting approximately 50% signaling activity in the basal state [52]. Qin et al. used X-ray crystallography to determine the GHSR structure upon binding to the inverse agonist PF-05190457 [48]. This inverse agonist occupies cavity I of the ligand-binding pocket, but does not utilize cavity II. In other words, unlike typical agonists and antagonists, PF-05190457 does not span the salt bridge. Instead, it coordinates vertically from the shallow region of the GHSR to its central region (Fig. 4a). These additional cavities are not found upon binding to antagonists or agonists. Additionally, PF-05190457 directly interacts with Trp276^6.48, preventing this amino acid from shifting to its active form, thereby diminishing receptor activity. Genetic analysis has shown that patients with familial short stature have mutations in GHSR. Among them, Phe279Leu [53] and Ala204Glu [54] attenuate the basal activity of GHSR.

1-4. The roles of ghrelin and anamorelin in inflammation and their therapeutic potential

Several studies exploring ghrelin’s roles in the pathogenesis of inflammation have demonstrated its anti-inflammatory effects (Fig. 5). Ghrelin and GHSR are expressed in human T lymphocytes and monocytes [55]. Ghrelin was shown to suppress the production of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in these cells [55]. Ghrelin also inhibited leptin-induced cytokine production [55]. Ghrelin administration to patients with chronic respiratory infections suppressed neutrophil-dominant airway inflammation [56], and it improved the postoperative clinical course of patients with esophageal cancer [57]. Ghrelin was found to suppress inflammatory responses by inhibiting the NF-κB signal pathway in TNF-α-activated human umbilical vein endothelial cells and a human acute monocytic leukemia cell line [58]. Ghrelin also reduced NF-κB signaling in IL-1β-stimulated human pulposus cells by inhibiting phosphorylation of the NF-κB inhibitor IκBα [59, 60].

Fig. 5  Ghrelin mitigates inflammation by suppressing NF-κB signaling

Several cytokines, including TNF-α and IL-1β, activate the NF-κB signaling pathway by phosphorylation of the NF-κB inhibitor IκBα, leading to its ubiquitin-dependent degradation. Activated NF-κB members translocate into the nucleus and induce transcription of inflammatory genes, including Nlrp3, Tnf-α, Il-1β, and Il-18. Ghrelin suppresses the NF-κB signaling pathway by inhibiting IκBα phosphorylation, resulting in anti-inflammatory activity. Ghrelin also inhibits the microglial production of cytokines such as TNF-α, IL-1β, and IL-6. Anamorelin appears to suppress inflammation in a GHSR-dependent manner.

The therapeutic potential of ghrelin in other inflammation-related disorders has been explored. Multiple sclerosis (MS) is an autoimmune neurological disorder characterized by central nervous system inflammation and subsequent demyelination and axonal degeneration [61]. MS is a female-dominant disease, and a national registration survey in Japan showed that its prevalence has been increasing to what is now more than 20,000 cases [62]. Animal models of experimental autoimmune encephalomyelitis (EAE) have been established to investigate MS pathogenesis and develop appropriate pharmacotherapy [63, 64]. Ghrelin administration alleviated demyelination and inflammatory cytokine production in the spinal cord, and reduced motor deficits in an animal model of EAE induced by myelin oligodendrocyte glycoprotein (MOG) [65-67]. In the spinal cord of rats with EAE, intraperitoneal ghrelin administration suppressed NF-κB, NLRP3 inflammasome pathways, and pyroptosis, the last of which is a caspase-dependent type of cell lysis accompanied by the release of inflammatory cytokines, including IL-1β and IL-18 [67, 68]. Ghrelin also suppressed microglial activation and reduced mRNA expression of IL-1β, IL-6, and TNF-α in EAE spinal cords [65, 67].

Anamorelin, an oral GHSR agonist, was launched in Japan in 2021 (under the brand name Adlumiz) for the treatment of cancer cachexia. Its molecular weight is 583 Da, and its chemical structure is based on the N-terminal active core of ghrelin [69, 70]. Anamorelin has a half-life of 7–12 hours, which is longer than that of ghrelin [69]. Cancer cachexia, a disorder characterized by rapid body weight loss with specific depletion of skeletal muscle and adipose tissue, is caused by tumor-derived catabolic factors and pro-inflammatory mediators arising from tumor-immune system crosstalk [71]. In patients with cancer cachexia, anamorelin improved daily activity and increased lean body mass and appetite [72]. We examined the therapeutic potential of anamorelin in a mouse model of MOG-induced EAE. Intraperitoneal anamorelin administration mitigated motor deficits and pathological alterations in EAE mice, and suppressed TNF-α and IFN-γ production in activated CD4⁺ lymphocytes in vitro. Ghrelin-based treatments could be used to augment immunosuppressive therapies currently used for inflammatory disorders, including MS.

2. LEAP2

2-1. The discovery of LEAP2

LEAP2 was originally isolated in 2003 from human plasma ultrafiltrate as a 40-amino acid basic peptide containing two disulfide bonds [73] (Fig. 1b). LEAP2 is cleaved from the 55-amino acid proLEAP2 by an endopeptidase targeted at typical Arg–Arg dibasic amino acids at positions 13 and 14 [73-75]. The LEAP2 sequence is identical between rodents and humans [74, 76]. As its name suggests, LEAP2 has antimicrobial activity against bacteria, and its production is induced following bacterial infection in immune tissues such as the liver, small intestine, bone marrow, and tonsils [73]. LEAP2 binds to the negatively charged surfaces of bacteria, permeabilizing their membranes and thus causing bacterial cell death [77]. This mechanism is common in antimicrobial peptides such as defensins [78, 79]. The portal vein allows direct transport of gut-derived products to the liver, and a bidirectional relationship between the gut and the liver is established by the liver feedback route of bile and antibody secretion to the intestine [80]. Tissues that have direct contact with the outer environment—such as the lungs, gastrointestinal tract, and skin—produce antimicrobial peptides that defend the body from exogenous microorganisms [81, 82]. The liver is thought to produce antimicrobial peptides to respond to the direct influx of microorganisms from the intestinal tract. LEAP2 does not affect monocyte chemotaxis or directly link innate and adaptive immunity. The concentrations at which LEAP2 exhibits antimicrobial activity are much higher than those observed physiologically; in humans, the effective antimicrobial concentration of LEAP2 (>6.6 μM = 30.2 μg/mL) is 3,000 times greater than its plasma concentration (~2 nM = 9.16 ng/mL) [83-86]. This suggests that LEAP2 may serve physiological functions beyond its antimicrobial activity.

Prior to the identification of LEAP2, LEAP1 was isolated from human plasma in 2,000 as an antimicrobial peptide mainly produced in the liver [84]. LEAP1, also named hepcidin, has been shown to play a role in iron metabolism [87, 88]. Hepcidin binds to ferroportin, an iron transport membrane protein that supplies intracellular iron to the blood [89]. The hepcidin expression level regulates the membrane secretory density of ferroportin [89].

2-2. GHSR as a receptor for LEAP2

In 2018, a research group in the United States demonstrated that LEAP2 is an endogenous peptide that is an antagonist of GHSR [83]. They explored substances whose expression levels were altered by vertical sleeve gastrectomy (VSG) in mice with diet-induced obesity (DIO). Leap2 expression was markedly upregulated after VSG surgery in the stomach, and was downregulated in the duodenum of mice fed a high-fat diet (HFD). The group studied both the agonist and antagonist activities of LEAP2 against 168 known human GPCRs [83]. As an antagonist, LEAP2 completely inhibited GHSR activity by a competitive ligand-binding assay with a SmBiT-based LEAP2 tracer [75]. LEAP2 alone did not modulate GHSR activity. LEAP2 functioned as a competitive antagonist when simultaneously administered with ghrelin because of its slow dissociation from GHSR, and as a non-competitive antagonist when added before ghrelin administration. The mode of LEAP2 action on GHSR varied under different experimental conditions.

In 2019, co-treatment of rat pancreatic islets with ghrelin and the 12-amino acid N-terminal fragment of LEAP2 showed that these amino acids attenuated the ghrelin-induced reduction of insulin levels in rat pancreatic islets [90]. Furthermore, ghrelin-induced food intake was inhibited in mice that were subcutaneously administered the 12-amino acid N-terminal fragment (0.6 nmol/g body weight (BW)), followed 10 minutes later by ghrelin (0.06 nmol/g BW) [90]. Another study showed that the 10-amino acid N-terminal fragment of LEAP2 bound to GHSR, thus suppressing insulin secretion from human pancreatic islets [91]. The researchers in that study demonstrated that LEAP2 expression was upregulated in enteroendocrine cells obtained during biopsy from patients with obesity [91]. We also showed that the 12-amino acid N-terminal fragment of LEAP2 inhibited ghrelin-induced GH secretion to the same extent as full-length LEAP2 when ghrelin and either LEAP2 or the N-terminal fragment of LEAP2 were administered to rat pituitary glands (unpublished data).

2-3. LEAP2 in metabolism

LEAP2 biosynthesis was studied under different nutritional conditions in mice [92]. The plasma LEAP2 concentration in DIO mice was higher than that in mice fed a chow diet, and was positively correlated with fat mass [92]. DIO reduced the plasma ghrelin concentration and increased the LEAP2/ghrelin ratio. Fasting decreased both the plasma LEAP2 concentration and the LEAP2/ghrelin ratio. Oral glucose administration to mice that had fasted for 24 hours increased the concentration of plasma LEAP2 and decreased that of ghrelin, while the plasma LEAP2 concentration was positively correlated with blood glucose concentration [92]. Compared with sham-operated mice fed a HFD, VSG mice fed a HFD had a lower Leap2 mRNA level in the liver and a lower plasma LEAP2 concentration [93]. A dietary change from a HFD to a chow diet in DIO mice also reduced both of these values. LEAP2 production is regulated by fat mass, food intake, and blood glucose.

The relationship between plasma LEAP2 and ghrelin was also studied in a cohort of adult men and women across several different body mass index (BMI) categories [19, 92]. The plasma LEAP2 concentration was positively correlated with BMI, percentage of body fat, fasting plasma glucose, fasting serum triglycerides, visceral adipose tissue volume, and intrahepatocellular lipid content measured by magnetic resonance spectroscopy [92, 94]. The postprandial plasma LEAP2 concentration was decreased after two types of weight loss surgery, specifically Roux-en-Y gastric bypass and VSG [92]. Serum LEAP2 concentrations were also measured in Japanese patients with obesity who underwent VSG [19]. The magnitudes of decreases in LEAP2 concentrations were positively correlated with decreases in body weight, visceral fat, and serum triglycerides after VSG, as well as with the excess weight loss rate, which represents postoperative efficacy [19]. The preoperative plasma LEAP2 concentration per body weight was positively correlated with postoperative weight loss effectiveness indices such as BMI, visceral fat area, serum triglycerides, hemoglobin A1c, and excess weight loss [19]. The preoperative plasma LEAP2 concentration may be a novel indicator in predicting weight loss after VSG surgery.

Leap2-deleted mice were developed in 2021 [95]. Leap2^–/– mice administered ghrelin (0.5–1.0 mg/kg BW) exhibited significant increases in 1- and 2-hour food intake compared with wild-type littermates given ghrelin [95]. Plasma GH concentrations were measured before and 15 minutes after the administration of ghrelin (0.1 mg/kg BW) [95]. The increase in the plasma GH concentration in Leap2^–/– mice was significantly greater than that in their wild-type littermates [95]. LEAP2 deletion in mice augmented ghrelin-induced food intake and GH secretion [95]. Histologically, there was significantly greater hepatic lipid accumulation in Leap2^–/– female DIO mice than in their wild-type DIO littermates [95]. Leap2^–/– female mice exhibited decreased energy expenditure and locomotor activity, and increased food intake [95]. By contrast, Leap2^–/– male mice did not demonstrate these phenotypes when fed either a HFD or a chow diet [95].

During glucose restriction, fatty acid-derived beta-hydroxybutyrate (BHB) is synthesized as an energy source in the liver [96]. Leap2 expression in the mouse liver was decreased and serum BHB levels were increased both after fasting and after 3-week exposure to a ketogenic diet [97]. Oral BHB administration to mice reduced hepatocyte Leap2 mRNA levels and plasma LEAP2 concentrations. In humans, endurance exercise (60 minutes of exercise on a bike ergometer) increased BHB concentrations and decreased those of plasma LEAP2 [97]. Ketogenesis may downregulate LEAP2 production by hunger signaling during energy deprivation [97].

A recent review reported an interaction between ghrelin and circadian rhythms [98]. Metabolic control is affected by dysregulation of circadian clock genes or zeitgebers. The suprachiasmatic nucleus (SCN) in the anterior hypothalamus is the master circadian regulator, and it shows high expression of Ghsr mRNA [99]. Ghrelin may influence the interaction between metabolism and circadian rhythms by directly acting on GHSR in the SCN [98]. LEAP2 may also be involved in the regulation of circadian biology via the ghrelin/GHSR system.

2-4. Effect of exogenous LEAP2 administration

Intraperitoneal LEAP2 administration to mice 10 minutes before ghrelin administration attenuated ghrelin-induced GH release in a dose-dependent manner [83]. LEAP2 monoclonal antibody administration to mice that had fasted for 24 hours increased both peak GH secretion and total GH release [83]. Pretreating mice with a high dose of LEAP2 (3 μmol/kg BW) completely abolished ghrelin-induced food intake [83].

Effects of LEAP2 administration on postprandial glucose metabolism and ad libitum food intake were investigated in 20 healthy men [100]. LEAP2 was continuously administered intravenously at 25 pmol/kg/min for 290 minutes, and blood samples were collected every 30 minutes. LEAP2 reduced GH concentrations, postprandial plasma glucose concentrations, and food intake compared with saline administration [100]. LEAP2 also increased postprandial insulin secretion and suppressed lipolysis [100]. No adverse reactions were reported during LEAP2 administration in this clinical study.

3. Ghrelin–LEAP2 interactions (Graphical Abstract)

Graphical Abstract Ghrelin–LEAP2 interactions along the stomach–liver axis

Ghrelin acts as an agonist of GHSR to induce GH secretion, food intake, adiposity, anti-inflammation and maintenance of blood glucose under caloric restriction, whereas LEAP2 acts as an antagonist or an inverse agonist of GHSR to suppress these ghrelin-induced effects.

3-1. Ghrelin suppresses LEAP2 expression in the liver

Ghrelin administration to fasted mice decreased plasma LEAP2 concentrations and Leap2 mRNA levels in the liver in a GHSR-dependent manner [23]. AMP-activated protein kinase (AMPK), a signal transduction molecule downstream of the GHSR signaling pathway, was shown to reduce lipogenesis and lipid accumulation [101, 102]. Compound C, an AMPK inhibitor, canceled ghrelin-induced AMPK phosphorylation and suppressed Leap2 expression in a murine hepatocyte cell line, whereas the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) downregulated Leap2 expression [23]. Ghrelin suppressed hepatic LEAP2 expression through an AMPK-dependent pathway.

Ghrelin administration in a chronic liver injury model in mice restored liver fibrogenesis by inhibiting the TGF-β1/SMAD3 and NF-κB pathways [103, 104]. Plasma LEAP2 was positively correlated with liver fat content and inversely correlated with plasma ghrelin levels in humans [95]. Hepatic Leap2 expression in mice that exhibited diet-induced steatosis was higher than that in mice fed a chow diet [105]. Leap2 knockdown in mice ameliorated steatosis via a lipolytic/lipogenic pathway and improved insulin sensitivity via IRS/AKT signaling [105]. In patients with nonalcoholic fatty liver disease, the liver fibrosis area was positively correlated with plasma LEAP2 and hepatic Leap2 expression, but negatively correlated with the plasma ghrelin concentration [106]. LEAP2 activated hepatic stellate cells, while ghrelin inhibited this activation by suppressing the fibrogenic cytokine TGF-β1 [106]. Ghrelin–LEAP2 interaction might be involved in the pathogenesis of liver fibrosis in patients with metabolic dysfunction-associated steatotic liver disease.

Polycystic ovary syndrome (PCOS) is characterized by irregular menstruation, amenorrhea, acne, hirsutism, and insulin resistance [107]. Plasma ghrelin and LEAP2 concentrations in PCOS were lower than those in healthy subjects, and LEAP2 was negatively correlated with insulin resistance, BMI, and the free androgen index [108]. Decreased LEAP2 concentrations or ghrelin–LEAP2 imbalances may be involved in insulin resistance in PCOS.

3-2. LEAP2 suppresses ghrelin-induced GH release

Ghrelin activates the phospholipase C (PLC)-inositol (1,4,5) triphosphate (IP3) pathway in somatotrophs, which promotes GH secretion [11, 109]. In LEAP2-overexpressing mice transfected with adeno-associated virus carrying the Leap2 gene, both ghrelin and GH concentrations under caloric restriction were lower than those in control mice [83]. Blood glucose concentrations in LEAP2-overexpressing mice continuously declined, eventually below viable levels, as caloric restriction persisted [83]. Using osmotic mini-pumps to continuously deliver GH to LEAP2-overexpressing mice under caloric restriction maintained stable blood glucose concentrations [83]. LEAP2-mediated GH reduction decreased fat consumption and increased fat storage, which may promote obesity.

Neudesin, a protein mainly secreted from adipose tissue and the brain, negatively impacts sympathetic activity and regulates the development of obesity and obesity-related metabolic dysfunction [110]. In patients with adult growth hormone deficiency, the plasma neudesin concentration and the LEAP2/ghrelin ratio were significantly higher than those in controls, while ghrelin concentrations were significantly lower; in addition, the concentration of plasma LEAP2 was correlated with that of plasma neudesin [111]. Ghrelin–LEAP2 interactions may be involved in metabolic dysfunction in GH deficiency.

3-3. LEAP2 suppresses the orexigenic effect of ghrelin

Ghrelin exerts its orexigenic effect by depolarizing NPY/AgRP neurons [11, 14, 112]. To explore the effects of LEAP2 and ghrelin on NPY/AgRP neurons, whole-cell patch-clamp recordings were performed in brain sections of NPY-humanized Renilla reniformis green fluorescent protein mice [92]. LEAP2 administration (100 nM) hyperpolarized these neurons, and coadministration of ghrelin (100 nM) abolished LEAP2-induced hyperpolarization. Subsequent LEAP2 administration canceled ghrelin-induced depolarization in these neurons [92]. Taken together, LEAP2 inhibited the ghrelin-induced activation of NPY/AgRP neurons. LEAP2 attenuates the constitutive activity of GHSR, hyperpolarizes NPY/AgRP neurons, and suppresses the orexigenic effects of ghrelin. It is unclear if LEAP2 is produced in the brain or if peripheral LEAP2 crosses the blood–brain barrier to reach the brain. Intracerebroventricular LEAP2 administration (1 nmol) to rats suppressed the orexigenic effect of ghrelin [23]. The peripheral administration of a higher dose of LEAP2 (15 nmol) suppressed ghrelin-induced food intake [23]. Future studies should determine how LEAP2 in the central nervous system regulates ghrelin functions.

3-4. LEAP2 antagonizes the insulinostatic effect of ghrelin

In pancreatic islet β cells, ghrelin activates GHSR that is coupled to G_αi2 proteins. G_αi2 proteins reduce cAMP production, activate voltage-gated K_v channels, repolarize cell membrane potential, inhibit Ca²⁺ influx, and consequently suppress insulin secretion [109, 113, 114]. LEAP2 administration to pancreatic islets obtained from patients undergoing Roux-en-Y gastric bypass surgery for obesity treatment abolished ghrelin’s inhibitory effect on insulin secretion and increased insulin secretion [91, 115-117]. Curiously, ghrelin administration to the same pancreatic islets as above did not result in an insulinostatic effect, but instead increased insulin secretion [91].

GHSR is also expressed in pancreatic polypeptide (PP)-producing cells [118]. Pancreatic PP, a 36-amino acid peptide, is a member of the NPY family that suppresses food intake [119-121]. Ghrelin dose-dependently inhibited PP release from mouse and rat islets [122, 123]. By contrast, LEAP2 administration to mice stimulated PP secretion [118, 124]. The plasma PP concentration in mice showed a positive correlation with both fat mass and the plasma LEAP2 concentration, and was increased by administration of a GHSR antagonist ([D-Lys³]-GHRP-6) [118]. Ghrelin and LEAP2 may also act on GHSR in PP cells and regulate its secretion.

3-5. Ghrelin and LEAP2 in bacterial infections

LEAP2 is upregulated in bacterial infections, which cause anorexia, body temperature elevation, and delayed gastric emptying [85]. Bacterial infections also reduce ghrelin levels and body weight [125]. These pathophysiological abnormalities may be caused by an elevated LEAP2/ghrelin ratio [126]. Ghrelin attenuates systemic inflammation by decreasing sympathetic tone and increasing parasympathetic tone [11]. Sepsis-induced sympathetic excitation promotes norepinephrine release from postganglionic sympathetic nerves, stimulates α_2A adrenergic receptors, and promotes TNFα production. Human LEAP2 concentrations were elevated in the cerebrospinal fluid of patients with acute bacterial meningitis and in the serum of patients with bacteremia [86]. The relationship between ghrelin and LEAP2 in inflammatory pathogenesis is an important topic for future research.

Conclusions

AgRP and α-MSH have been identified as bioactive peptides that act as an agonist and antagonist, respectively, at the identical receptor, melanocortin-4 receptor (MC4R). Ghrelin and LEAP2 constitute a second combination acting at another identical receptor, GHSR. Ghrelin and LEAP2 regulate body weight, food intake, exercise, GH and blood glucose concentrations, and other physiological phenomena. A significant amount of research now focuses on ghrelin and LEAP2, and may uncover their therapeutic potential for the treatment of metabolic diseases. Further exploratory studies of ghrelin–LEAP2 interactions may identify unknown biological regulatory mechanisms and establish novel therapeutic approaches.

Author Contributions

K.S. wrote all sections other than, 1-2, 1-3, and 1-4. Y.S., W.Z., and Y.N. wrote sections (1-1, 1-3), 1-2, and 1-4, respectively. M.N. supervised the writing of all sections.

Disclosure

None of the authors have any potential conflicts of interest associated with this research.

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