Liver Expressed Antimicrobial Peptide 2 is Associated with Steatosis in Mice and Humans

Abstract

Background and Aims Liver expressed antimicrobial peptide 2 (LEAP2) is recently identified as a regulator in energy metabolism. This study aims to 1) investigate the role of leap2 in hepatic steatosis in C57BL/6 mice; 2) evaluate the association between circulating LEAP2 levels and liver fat contents in a hospital based case-control study.

Methods The rodent experiment: western blotting and qPCR were performed to evaluate leap2 levels, lipid metabolism pathways and insulin signaling. shRNA was used to knockdown leap2. The clinical study: commercial ELISA kits were used to measure circulating LEAP2 levels (validated by western blotting). Liver fat content was estimated using MRI-derived proton density fat fraction and FibroScan-derived controlled attenuation parameter.

Results The rodent experiment found the hepatic expression and secreted levels of leap2 were increased in mice with diet-induced steatosis. Leap2 knockdown ameliorated steatosis via lipolytic/lipogenic pathway and improved insulin sensitivity via IRS/AKT signaling. The clinical study reported increased circulating levels of LEAP2 in the subjects with steatosis. Moreover, LEAP2 correlated positively with age, body mass index, waist-to-hip ratio, liver fat content, fasting insulin and HOMA-IR, whereas inversely with acyl-ghrelin. Furthermore, the circulating levels of LEAP2 are dependent on liver fat content, acyl-ghrelin and fasting glucose. Lastly, circulating LEAP2 is an independent predictor of NAFLD.

Conclusions The study suggests LEAP2 is associated with hepatic steatosis, which may involve lipolytic/lipogenic pathway and insulin signaling.

Key words

controlled attenuation parameter - liver expressed antimicrobial peptide 2 - magnetic resonance imaging - nonalcoholic fatty liver disease - proton density fat fraction

Publication History

Received: 05 May 2020
Received: 13 June 2020

Accepted: 30 June 2020

Article published online:
15 September 2020

• References

• 1 Marchesini G, Petta S, Dalle Grave R. Diet, weight loss, and liver health in nonalcoholic fatty liver disease: Pathophysiology, evidence, and practice. Hepatology 2016; 63: 2032-2043 DOI: 10.1002/hep.28392
• 2 Chalasani N, Younossi Z, Lavine JE. et al. The diagnosis and management of non-alcoholic fatty liver disease: Practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology 2012; 55: 2005-2023 doi:10.1002/hep.25762
• 3 Birkenfeld AL, Shulman GI. Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. Hepatology 2014; 59: 713-723 DOI: 10.1002/hep.26672
• 4 Krause A, Sillard R, Kleemeier B. et al. Isolation and biochemical characterization of LEAP-2, a novel blood peptide expressed in the liver. Protein Sci 2003; 12: 143-152 doi:10.1110/ps.0213603
• 5 Henriques ST, Tan CC, Craik DJ. et al. Structural and functional analysis of human liver-expressed antimicrobial peptide 2. Chembiochem 2010; 11: 2148-2157 doi:10.1002/cbic.201000400
• 6 Howard A, Townes C, Milona P. et al. Expression and functional analyses of liver expressed antimicrobial peptide-2 (LEAP-2) variant forms in human tissues. Cell Immunol 2010; 261: 128-133 doi:10.1016/j.cellimm.2009.11.010
• 7 Ge X, Yang H, Bednarek MA. et al. LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor. Cell Metab 2018; 27: 461-469.e466 doi:10.1016/j.cmet.2017.10.016
• 8 M'Kadmi C, Cabral A, Barrile F. et al. N-Terminal Liver-Expressed Antimicrobial Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the Ghrelin Receptor. J Med Chem 2019; 62: 965-973 doi:10.1021/acs.jmedchem.8b01644
• 9 Mani BK, Puzziferri N, He Z. et al. LEAP2 changes with body mass and food intake in humans and mice. J Clin Invest 2019; 129: 3909-3923 doi:10.1172/jci125332
• 10 Wang JH, Li HZ, Shao XX. et al. Identifying the binding mechanism of LEAP2 to receptor GHSR1a. Febs J 2019; 286: 1332-1345 doi:10.1111/febs.14763
• 11 Abizaid A, Hougland JL. Ghrelin Signaling: GOAT and GHS-R1a Take a LEAP in Complexity. Trends Endocrinol Metab 2020; 31: 107-117 DOI: 10.1016/j.tem.2019.09.006
• 12 Andrews ZB. The next big LEAP2 understanding ghrelin function. J Clin Invest 2019; 129: 3542-3544 DOI: 10.1172/jci131023
• 13 Schalla MA, Stengel A. LEAP2: A novel regulator of food intake and body weight?. Nat Rev Gastroenterol Hepatol 2019; 16: 711-712 DOI: 10.1038/s41575-019-0224-9
• 14 Zhou W, Yang J, Zhu J. et al. Fetuin B aggravates liver X receptor-mediated hepatic steatosis through AMPK in HepG2 cells and mice. Am J Transl Res 2019; 11: 1498-1509
• 15 Yang J, Zhou W, Zhu J. et al. Circulating ectodysplasin A is a potential biomarker for nonalcoholic fatty liver disease. Clin Chim Acta 2019; 499: 134-141 doi:10.1016/j.cca.2019.09.009
• 16 Zhu J, Wan X, Wang Y. et al. Serum fetuin B level increased in subjects of nonalcoholic fatty liver disease: a case-control study. Endocrine 2017; 56: 208-211 doi:10.1007/s12020-016-1112-5
• 17 Attard SM, Howard AG, Herring AH. et al. Differential associations of urbanicity and income with physical activity in adults in urbanizing China: findings from the population-based China Health and Nutrition Survey 1991-2009. Int J Behav Nutr Phys Act 2015; 12: 152 doi:10.1186/s12966-015-0321-2
• 18 Ainsworth BE, Haskell WL, Herrmann SD. et al. 2011 Compendium of Physical Activities: a second update of codes and MET values. Med Sci Sports Exerc 2011; 43: 1575-1581 doi:10.1249/MSS.0b013e31821ece12
• 19 Chobanian AV, Bakris GL, Black HR et al. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension 2003; 42: 1206–1252
• 20 Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: Diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med 1998; 15: 539-553 DOI: 10.1002/(SICI)1096-9136(199807)15:7<539::AID-DIA668>3.0.CO;2-S
• 21 Lin SC, Heba E, Wolfson T. et al. Noninvasive Diagnosis of Nonalcoholic Fatty Liver Disease and Quantification of Liver Fat Using a New Quantitative Ultrasound Technique. Clin Gastroenterol Hepatol 2015; 13: 1337-1345 e1336 doi:10.1016/j.cgh.2014.11.027
• 22 Caussy C, Alquiraish MH, Nguyen P. et al. Optimal threshold of controlled attenuation parameter with MRI-PDFF as the gold standard for the detection of hepatic steatosis. Hepatology 2018; 67: 1348-1359 doi:10.1002/hep.29639
• 23 Sasso M, Tengher-Barna I, Ziol M. et al. Novel controlled attenuation parameter for noninvasive assessment of steatosis using Fibroscan((R)): Validation in chronic hepatitis C. J Viral Hepat 2012; 19: 244-253 doi:10.1111/j.1365-2893.2011.01534.x
• 24 Bedogni G, Bellentani S, Miglioli L. et al. The Fatty Liver Index: A simple and accurate predictor of hepatic steatosis in the general population. BMC Gastroenterol 2006; 6: 33. doi:10.1186/1471-230X-6-33
• 25 Zhu J, He M, Zhang Y. et al. Validation of simple indexes for nonalcoholic fatty liver disease in western China: A retrospective cross-sectional study. Endocrine journal. 2018; DOI: 10.1507/endocrj.EJ17-0466
• 26 Faul F, Erdfelder E, Buchner A. et al. Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behavior research methods 2009; 41: 1149-1160 doi:10.3758/BRM.41.4.1149
• 27 Al-Massadi O, Muller T, Tschop M. et al. Ghrelin and LEAP-2: Rivals in Energy Metabolism. Trends Pharmacol Sci 2018; 39: 685-694 doi:10.1016/j.tips.2018.06.004
• 28 Islam MN, Mita Y, Maruyama K. et al. Liver-expressed antimicrobial peptide 2 antagonizes the effect of ghrelin in rodents. J Endocrinol 2020; 244: 13-23 doi:10.1530/joe-19-0102
• 29 Alves-Bezerra M, Cohen DE. Triglyceride Metabolism in the Liver. Compr Physiol 2017; 8: 1-8 DOI: 10.1002/cphy.c170012
• 30 Montagner A, Polizzi A, Fouche E. et al. Liver PPARalpha is crucial for whole-body fatty acid homeostasis and is protective against NAFLD. Gut 2016; 65: 1202-1214 doi:10.1136/gutjnl-2015-310798
• 31 Wang Y, Viscarra J, Kim SJ. et al. Transcriptional regulation of hepatic lipogenesis. Nature Reviews Molecular Cell Biology 2015; 16: 678-689 doi:10.1038/nrm4074
• 32 Donnelly KL, Smith CI, Schwarzenberg SJ. et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. The Journal of Clinical Investigation 2005; 115: 1343-1351 doi:10.1172/JCI23621
• 33 Ducheix S, Montagner A, Theodorou V. et al. The liver X receptor: A master regulator of the gut-liver axis and a target for non alcoholic fatty liver disease. Biochemical Pharmacology 2013; 86: 96-105 doi:10.1016/j.bcp.2013.03.016
• 34 Joseph SB, Laffitte BA, Patel PH. et al. Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors. The Journal of biological chemistry 2002; 277: 11019-11025 doi:10.1074/jbc.M111041200
• 35 Talukdar S, Hillgartner FB. The mechanism mediating the activation of acetyl-coenzyme A carboxylase-alpha gene transcription by the liver X receptor agonist T0-901317. Journal of Lipid Research 2006; 47: 2451-2461 DOI: 10.1194/jlr.M600276-JLR200
• 36 Chu K, Miyazaki M, Man WC. et al. Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation. Molecular and Cellular Biology 2006; 26: 6786-6798 doi:10.1128/MCB.00077-06
• 37 Repa JJ, Liang G, Ou J. et al. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta. Genes & Development 2000; 14: 2819-2830
• 38 Liang G, Yang J, Horton JD. et al. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. The Journal of Biological Chemistry 2002; 277: 9520-9528 doi:10.1074/jbc.M111421200
• 39 Bhattacharyya S, Feferman L, Tobacman JK. Carrageenan Inhibits Insulin Signaling through GRB10-mediated Decrease in Tyr(P)-IRS1 and through Inflammation-induced Increase in Ser(P)307-IRS1. J Biol Chem 2015; 290: 10764-10774 DOI: 10.1074/jbc.M114.630053
• 40 Ke B, Zhao Z, Ye X. et al. Inactivation of NF-kappaB p65 (RelA) in Liver Improves Insulin Sensitivity and Inhibits cAMP/PKA Pathway. Diabetes 2015; 64: 3355-3362 doi:10.2337/db15-0242
• 41 Fittipaldi AS, Hernandez J, Castrogiovanni D. et al. Plasma levels of ghrelin, des-acyl ghrelin and LEAP2 in children with obesity: correlation with age and insulin resistance. Eur J Endocrinol 2020; 182: 165-175 doi:10.1530/eje-19-0684
• 42 Aslanipour B, Alan M, Demir I. Decreased levels of liver-expressed antimicrobial peptide-2 and ghrelin are related to insulin resistance in women with polycystic ovary syndrome. Gynecol Endocrinol 2019; 1-4 DOI: 10.1080/09513590.2019.1665646
• 43 Reeder SB, Cruite I, Hamilton G. et al. Quantitative Assessment of Liver Fat with Magnetic Resonance Imaging and Spectroscopy. J Magn Reson Imaging 2011; 34: 729-749 doi:10.1002/jmri.22580 10.1002/jmri.22775
• 44 Fishbein M, Castro F, Cheruku S. et al. Hepatic MRI for fat quantitation: its relationship to fat morphology, diagnosis, and ultrasound. J Clin Gastroenterol 2005; 39: 619-625 doi:10.1097/00004836-200508000-00012
• 45 Boursier J, Cales P. Controlled attenuation parameter (CAP): A new device for fast evaluation of liver fat?. Liver Int 2012; 32: 875-877 DOI: 10.1111/j.1478-3231.2012.02824.x