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LEAP-2 produced in the small intestine modulates Ghrelin function in response to fasting and food intake

leap 2 precursor structure

sequence comparison leap 2

trypsin digestion of LEAP-2

Human LEAP-2 precursor cDNA and deduced amino acid sequence. Human LEAP-2 precursor cDNA and deduced amino acid sequence. Coding regions are printed in capital letters. The typical secretory signal sequence 1–22 is printed in italics, whereas the positions of introns 1 (198 bp) and 2 (320 bp) of the LEAP-2 gene are marked by vertical lines. Two TATAAA sequences located 31 and 84 bases upstream of the translational start were identified. The putatively mature and antimicrobially active LEAP-2-(38–77) isolated from hemofiltrate and the putative polyadenylation signal are underlined. The position of the primers used for nested 5'-RACE-PCR and for preparative PCR from six different species is indicated.


Alignment of LEAP-2 from mammalian species.

Alignment of LEAP-2 from mammalian species. Standard PCR carried out with primers originally designed for the human LEAP-2 gene revealed homologous LEAP-2 forms in rhesus monkey, cow, pig, mouse, and guinea pig. The depicted putative peptide sequences are deduced from the cDNA sequences obtained. Underlined amino acids represent the putative signal peptide sequences as predicted by the SignalP V2.0 program (Nielsen et al. 1997), and the mature LEAP-2 (38–77) form is hyphenated. The nucleotide sequence data reported in this paper have been submitted to the GenBank/EBI Data Bank with accession numbers AJ306405 (Homo sapiens mRNA), AJ409013 (Sus scrofa mRNA), AJ409014 (Bos taurus mRNA), AJ409054 (Cavia porcellus mRNA), AJ409055 (Mus musculus mRNA), AJ409056 (Macaca mulatta genomic DNA), AJ409063 (Mus musculus genomic DNA), AJ409064 (Homo sapiens genomic DNA), and AJ409065 (Homo sapiens alternative promoter sequence).

Context: The mechanisms underlying Roux-en-Y gastric bypass (RYGB) surgery-induced weight loss and the immediate postoperative beneficial metabolic effects associated with the operation remain uncertain. Enteroendocrine cell (EEC) secretory function has been proposed as a key factor in the marked metabolic benefits from RYGB surgery.

Objective: To identify novel gut-derived peptides with therapeutic potential in obesity and/or diabetes by profiling of EEC-specific molecular changes in obese patients following RYGB-induced weight loss.

Subjects and methods: Genome-wide expression analysis was performed in isolated human small intestinal EECs obtained from 20 gut-biopsied obese subjects before and after RYGB. Targets of interest were profiled for preclinical and clinical metabolic effects.

Results: RYGB consistently increased expression levels of the inverse ghrelin receptor agonist, liver-expressed antimicrobial peptide 2 (LEAP2). A secreted endogenous LEAP2 fragment (LEAP238-47) demonstrated robust insulinotropic properties, stimulating insulin release in human pancreatic islets comparable to the gut hormone glucagon-like peptide-1. LEAP238-47 showed reciprocal effects on growth hormone secretagogue receptor (GHSR) activity, suggesting that insulinotropic action of the peptide may be directly linked to attenuation of tonic GHSR activity. The fragment was infused in healthy human individuals (n=10), but no glucoregulatory effect was observed in the chosen dose as compared to placebo.

Conclusions: Small intestinal LEAP2 expression was upregulated after RYGB. The corresponding circulating LEAP238-47 fragment demonstrated strong insulinotropic action in vitro, but failed to elicit glucoregulatory effects in healthy human subjects.

Hagemann CA, Zhang C, Hansen HH, et al. Identification and metabolic profiling of a novel human gut-derived LEAP2 fragment. The Journal of Clinical Endocrinology & Metabolism

Background/objectives: Liver-expressed antimicrobial peptide 2 (LEAP-2) was recently identified as an endogenous non-competitive allosteric antagonist of the growth hormone secretagogue receptor 1a (GHSR1a). LEAP-2 blunts ghrelin-induced feeding and its plasma levels are modulated in response to nutritional status in humans. Despite the relevant role of ghrelin in childhood, puberty, and childhood obesity, the potential implication of LEAP-2 in these aspects remains totally unknown. We aimed to investigate the regulation of circulating plasma LEAP-2 in childhood and adolescent either lean or obese.

Methods and results: Plasma levels of LEAP-2 were analyzed in a cross-sectional study with lean and obese children and adolescents (n = 150). Circulating LEAP-2 levels were significantly higher in girls than in boys independently of whether they were obese or lean. In addition, LEAP-2 was significantly increased (p < 0.001) in pubertal than in prepubertal girls, while no changes were found in boys between both developmental stages. Moreover, in girls LEAP-2 was positively correlated with insulin, IGF-1, HOMA-IR and triglycerides and negatively with ghrelin. In boys, LEAP-2 was positively correlated with leptin and negatively with vitamin D levels.

Conclusion: This study reveals a sexual dimorphism in LEAP-2 levels in children and adolescents. These changes and the higher levels during puberty imply that LEAP-2 may contribute to some of the biological adaptations occurring during pubertal development in terms of food intake, energy balance, growth rate, and puberty onset. Future studies assessing LEAP-2 levels in longitudinal studies and its implications in growth rate, puberty onset, and reproductive hormones will help to understand the relevance of this hormone in this stage of life.


Barja-Fernández S, Lugilde J, Castelao C, et al. Circulating LEAP-2 is associated with puberty in girls. Int J Obes. 

Acyl-ghrelin administration increases food intake, body weight, and blood glucose. In contrast, mice lacking ghrelin or ghrelin receptors (GHSRs) exhibit life-threatening hypoglycemia during starvation-like conditions, but do not consistently exhibit overt metabolic phenotypes when given ad libitum food access. These results, and findings of ghrelin resistance in obese states, imply nutritional state dependence of ghrelin’s metabolic actions. Here, we hypothesized that liver-enriched antimicrobial peptide-2 (LEAP2), a recently characterized endogenous GHSR antagonist, blunts ghrelin action during obese states and postprandially. To test this hypothesis, we determined changes in plasma LEAP2 and acyl-ghrelin due to fasting, eating, obesity, Roux-en-Y gastric bypass (RYGB), vertical sleeve gastrectomy (VSG), oral glucose administration, and type 1 diabetes mellitus (T1DM) using humans and/or mice. Our results suggest that plasma LEAP2 is regulated by metabolic status: its levels increased with body mass and blood glucose and decreased with fasting, RYGB, and in postprandial states following VSG. These changes were mostly opposite of those of acyl-ghrelin. Furthermore, using electrophysiology, we showed that LEAP2 both hyperpolarizes and prevents acyl-ghrelin from activating arcuate NPY neurons. We predict that the plasma LEAP2/acyl-ghrelin molar ratio may be a key determinant modulating acyl-ghrelin activity in response to body mass, feeding status, and blood glucose.


Mani BK, Puzziferri N, He Z, et al. LEAP2 changes with body mass and food intake in humans and mice. Journal of Clinical Investigation. 2019;129(9):3909-3923.

Rheumatoid arthritis (RA) is a debilitating, chronic, inflammatory, autoimmune disease associated with cachexia. The substitutive therapy of gut hormone ghrelin has been pointed at as a potential countermeasure for the management of metabolic and inflammatory complications in RA. The recent discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous inverse agonist/antagonist of the ghrelin receptor makes feasible the development of a more rational pharmacological approach. This work aimed to assess the serum LEAP2 levels, in a cohort of RA patients, in comparison with healthy individuals and determine its correlation with inflammatory parameters. LEAP2 levels were determined by a commercial ELISA kit, plasma C-reactive protein (CRP) levels were evaluated using immunoturbidimetry, and serum levels of inflammatory mediators, namely IL-6, IL-8, IL-1β, MIP1α, MCP1, and LCN2, were measured by XMap multiplex assay. LEAP2 serum levels were significantly increased in RA patients (n = 101) compared with control subjects (n = 26). Furthermore, the LEAP2 levels significantly correlated with CRP and inflammatory cytokines, but not with BMI. These data reveal LEAP2 as a new potential RA biomarker and indicated the pharmacological control of LEAP2 levels as a novel approach for the treatment of diseases with alterations on the ghrelin levels, such as rheumatoid cachexia.


Francisco V, Tovar S, Conde J, et al. Levels of the novel endogenous antagonist of ghrelin receptor, liver-enriched antimicrobial peptide-2, in patients with rheumatoid arthritis. Nutrients. 2020;12(4):1006.

Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo, including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo. Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.

Xuecai Ge, Hong Yang, Maria A. Bednarek et al., Cell Metabolism 2018 ; Published online: December 7, 201

Interleukin-1β (IL-1β), an inflammatory cytokine of the IL-1 family, is primarily produced as a precursor protein by monocytes and macrophages, then matures and becomes activated through proteolytic catalysis. Although the biological characteristics of avian IL-1β are well known, little information is available about its biological role in songbird species such as house finches that are vulnerable to naturally-occurring inflammatory diseases. In this study, house finch IL-1β (HfIL-1β) was cloned, expressed, and its biological function examined. Both precursor and mature forms of HfIL-1β consisting of 269 and 162 amino acids, respectively, were amplified from total RNA of spleen and cloned into expression vectors. HfIL-1β showed high sequential and tertiary structural similarity to chicken homologue that allowed detection of the expressed mature recombinant HfIL-1β (rHfIL-1β) with anti-ChIL-1β antibody by immunoblot analysis. For further characterization, we used primary splenocytes and hepatocytes that are predominant sources of IL-1β upon stimulation, as well as suitable targets to stimulation by IL-1β. Isolated house finch splenocytes were stimulated with rHfIL-1β in the presence and absence of concanavalin A (Con A), RNA was extracted and transcript levels of Th1/Th2 cytokines and a chemokine were measured by qRT-PCR. The addition of rHfIL-1β induced significant enhancement of IL-2 transcript, a Th1 cytokine, while transcription of IL-1β and the Th2 cytokine IL-10 was slightly enhanced by rHfIL-1β treatment. rHfIL-1β also led to elevated levels of the chemokine CXCL1 and nitric oxide production regardless of co-stimulation with Con A. In addition, the production of the acute phase protein serum amyloid A and the antimicrobial peptide LEAP2 was observed in HfIL-1β-stimulated hepatocytes. Taken together, these observations revealed the basic functions of HfIL-1β including the stimulatory effect on cell proliferation, production of Th1/Th2 cytokines and acute phase proteins by immune cells, thus providing valuable insight into how HfIL-1β is involved in regulating inflammatory response.

Park M, Kim S, Adelman JS, Leon AE, Hawley DM, Dalloul RA. Identification and functional characterization of the house finch interleukin-1β. Dev Comp Immunol. 2017;69:41-50.

Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to its mammalian orthologues at both structural and sequence levels. The gene is expressed in the embryo mostly in the endoderm-derived tissues. Accordingly it is induced in pluripotent animal cap cells by FGF, activin or a combination of vegT/?-catenin. Modulating leap2 expression level by gain-of-function strategy impaired normal embryonic development. When overexpressed in pluripotent embryonic cells derived from blastula animal cap explant, leap2 stimulated FGF while it reduced the activin response . Finally, we demonstrate that LEAP2 blocks FGF-induced migration of HUman Vascular Endothelial Cells (HUVEC). Altogether these findings suggest a model in which LEAP2 could act at the extracellular level as a modulator of FGF and activin signals, thus opening new avenues to explore it in relation with cellular processes such as cell differentiation and migration.

Thiébaud P, Garbay B, Auguste P, et al. Overexpression of Leap2 impairs Xenopus embryonic development and modulates FGF and activin signals. Peptides. 2016;83:21-8.

BACKGROUND: Immune activation is one of the main features of HIV/Hepatitis C virus (HCV) infections and has been linked to the disturbance of the gut-associated lymphoid tissue (GALT). In chronic HIV infection, loss of GALT integrity results in translocation of microbial products and chronic immune activation. We explored the relationship between bacterial translocation and specific colonic proteins, including liver expressed antimicrobial peptide (LEAP 2) which may play a role in modulating the bacterial translocation process.
METHODS: A total of 40 subjects (10 HIV/HCV, 10 HIV, 10 HCV-infected patients and 10 controls) were enrolled and underwent serum and colonic tissue sampling. The levels of immune activation were evaluated by measuring plasma sCD27, and the levels of choosen proinflammatory, Th2 and regulatory cytokines in both the plasma and supernatant of CD3-stimulated intraepithelial lymphocytes. We also evaluated LEAP-2 expression in the colon biopsies using Affymetrix Human Gene 1.0 ST (HuGene) and fluorescent immunohistochemistry.
RESULTS: Increased levels of sCD27 were observed in HIV/HCV coinfected (p=0.03) and HIV monoinfected (p=0.04) patients compared with controls consistent with the presence of immune activation. The chip array identified LEAP-2 expression as a key marker associated with immune activation. LEAP-2 expression in HIV, HCV and HIV/HCV-infected patients was significantly lower compared with controls, and was significantly negatively correlated (p=0.03, r=-0.44) with sCD27.
CONCLUSIONS: Our data suggests that HCV and HIV infections are associated with decreased expression of LEAP-2 in colonic tissue. This may represent a key mechanism for enhanced microbial translocation and immune activation in HIV/HCV-infected patients.
This publication used a LEAP-2 antibody (#H-075-40) from Phoenix Pharmaceuticals.
Shata MT, Abdel-hameed EA, Hetta HF, Sherman KE. Immune activation in HIV/HCV-infected patients is associated with low-level expression of liver expressed antimicrobial peptide-2 (LEAP-2). J Clin Pathol. 2013;66(11):967-75.

LEAP-2 Levels in human plasma before and after feeding (Measured Via EIA & RIA)

measured level before after mealmeasured level before after meal EIA

Human serum samples were assayed using our internally developed LEAP-2 (38-77) EIA kit (#EK-075-40). All samples were assayed in duplicate. On average, LEAP-2 levels increased by 21% (6.54 ng/ml vs. 7.92 ng/ml) after feeding (compared to overnight fasting).

 

measured level before after meal RIAmeasured level before after meal RIA

Human plasma samples were assayed using our internally developed LEAP-2 (38-77) RIA kit (#RK-075-40). All samples were assayed in duplicate. On average, LEAP-2 levels increased by 21% (7.34 ng/ml vs. 8.67 ng/ml) after feeding (compared to overnight fasting).


 

Ghrelin maintains blood glucose levels in the face of starvation and LEAP-2 Peptide modulates ghrelin function in response to nutrient status, such as fasting          

The figure demonstrates that Ghrelin maintains blood glucose levels in the face of starvation and LEAP-2 Peptide modulates ghrelin function in response to nutrient status, such as fasting. 
Increasing or suppressing LEAP2 leads to the corresponding counter-regulation of ghrelin action in vivo.
Figure from Xuecai Ge et al. , LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor. Cell Metabolism Published online: December 7, 2017

 

expression of LEAP-2 protein in the colonic tissue
comparative evaluation of LEAP-2 expression in colonic tissues from a healthy control subject and HIV-monoinfected patients.
Expression of LEAP-2 protein in the colonic tissue was evaluated using fluorescent immunohistochemistry. (A) Represents the expression of LEAP-2 protein in colonic tissue from normal healthy control subject using anti-LEAP-2 antibody (A2), positive control colon biopsy samples from a patient with Crohn’s disease (A3) or patient with ulcerative colitis (A4) or isotype control (A1) and the secondary antibody Alexa Fluor 594 donkey anti-rabbit IgG. LEAP-2 appears as red particles in intraepithelial and interstitial tissue of the colon. (B) Shows representatives data of the corresponding haematoxylin and eosin (H&E) staining for colonic tissues of the subjects demonstrated an increase in infiltrating inflammatory cells (markers of immune activation)

Alternative splicing in the LEAP-2 gene. Alternative splicing in the LEAP-2 gene. (A) Standard PCR conducted with the primer combination E1-S and E3-AS (Fig. 2) and 20 ng of DNase-treated cDNA. LEAP-2350 is mainly expressed in liver, kidney, and colon, whereas LEAP-2550 is the main transcript in lung, trachea, and heart. PCR products at 650 bp, 720 bp, and 870 bp represent splicing variants of LEAP-2, and the band at 480 bp could be identified as a PCR artefact. (MW = marker, 100 bp DNA ladder, Life Technologies). (B) Standard PCR performed with the primers PROM-S (5'-GGTGCA GATTAGGGTGACAGTCCATC-3'), which is located 565 bp upstream of the transcriptional start depicted in Figure 2, and E3-AS. Lung, heart, and trachea exhibit the same pattern of bands, including the 565 bp shift in size caused by the upstream primer PROM-S. The main transcript identified in liver, kidney, and colon, however, changes from the completely spliced LEAP-2350 to the intron 1-retaining LEAP-2 variant. All bands obtained were characterized by DNA sequencing.
Northern Blot analysis. Northern Blot analysis. Human MTN Blots I+II (Clontech, A+B) were hybridized under high-stringency conditions with a 32P-labeled LEAP-2-specific cDNA fragment. A transcript size of 0.7 kb identified in liver, kidney, and small intestine represents the complete LEAP-2 cDNA including a poly(A)-tail of 200 bp. The signal at 2.0 kb corresponds to the LEAP-2 form coded by the distal promoter, which could also be identified in 5'-RACE-PCR, whereas the bands at 4.2 kb and 8.0 kb might either represent additional alternative promoter variants or homologous proteins related to LEAP-2.
Antimicrobial activity of LEAP-2-(38–77) and LEAP-2-(44–77). Antimicrobial activity of LEAP-2-(38–77) and LEAP-2-(44–77). Colony-forming unit assay of synthetic LEAP-2-(38–77) (solid triangles) and LEAP-2-(44–77) (open circles), the two main peptide forms isolated from hemofiltrate, against S. cerevisiae ATCC9763. Incubation without peptide represents 100% CFU. Synthetic and native LEAP-2-(44–77) led to similar results. The bars indicate the minimum and maximum value of the triplicates used in this representative assay. The inset depicts the dose-dependent effect of LEAP-2-(38–77) against S. cerevisiae in a radial diffusion assay. LEAP-2-(44–77) showed no effect in this sensitive antimicrobial assay. The antimicrobially active casocidin-I (11 µg/well) served as a positive control ( Zucht et al. 1998). One inhibition unit (1 IU) corresponds to 0.1 mm diameter of growth inhibition zone, and the error bars represent the S.D. calculated from three experiments performed.



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