Advertisement

Metabolomic Identification of Subtypes of Nonalcoholic Steatohepatitis

Published:January 26, 2017DOI:https://doi.org/10.1053/j.gastro.2017.01.015

      Background & Aims

      Nonalcoholic fatty liver disease (NAFLD) is a consequence of defects in diverse metabolic pathways that involve hepatic accumulation of triglycerides. Features of these aberrations might determine whether NAFLD progresses to nonalcoholic steatohepatitis (NASH). We investigated whether the diverse defects observed in patients with NAFLD are caused by different NAFLD subtypes with specific serum metabolomic profiles, and whether these can distinguish patients with NASH from patients with simple steatosis.

      Methods

      We collected liver and serum from methionine adenosyltransferase 1a knockout (MAT1A-KO) mice, which have chronically low levels of hepatic S-adenosylmethionine (SAMe) and spontaneously develop steatohepatitis, as well as C57Bl/6 mice (controls); the metabolomes of all samples were determined. We also analyzed serum metabolomes of 535 patients with biopsy-proven NAFLD (353 with simple steatosis and 182 with NASH) and compared them with serum metabolomes of mice. MAT1A-KO mice were also given SAMe (30 mg/kg/day for 8 weeks); liver samples were collected and analyzed histologically for steatohepatitis.

      Results

      Livers of MAT1A-KO mice were characterized by high levels of triglycerides, diglycerides, fatty acids, ceramides, and oxidized fatty acids, as well as low levels of SAMe and downstream metabolites. There was a correlation between liver and serum metabolomes. We identified a serum metabolomic signature associated with MAT1A-KO mice that also was present in 49% of the patients; based on this signature, we identified 2 NAFLD subtypes. We identified specific panels of markers that could distinguish patients with NASH from patients with simple steatosis for each subtype of NAFLD. Administration of SAMe reduced features of steatohepatitis in MAT1A-KO mice.

      Conclusions

      In an analysis of serum metabolomes of patients with NAFLD and MAT1A-KO mice with steatohepatitis, we identified 2 major subtypes of NAFLD and markers that differentiate steatosis from NASH in each subtype. These might be used to monitor disease progression and identify therapeutic targets for patients.

      Keywords

      Abbreviations used in this paper:

      ALT (alanine aminotransferase), DG (diglycerides), DMR (differentially methylated DNA regions), FA (fatty acids), GSH (glutathione), JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl-carbocyanineiodide), LC (liquid chromatography), MAT1A-KO (methionine adenosyltransferase 1a knockout), MS (mass spectrometry), NAFLD (nonalcoholic fatty liver disease), NASH (nonalcoholic steatohepatitis), PC (phosphatidylcholine), PCA (principal component analysis), PE (phosphatidylethanolamine), SAMe (S-adenosylmethionine), TG (triglycerides), VLDL (very-low-density lipoprotein), WT (wild-type)
      To read this article in full you will need to make a payment
      AGA Member Login
      Login with your AGA username and password.
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Cohen J.C.
        • Horton J.D.
        • Hobbs H.H.
        Human fatty liver disease: old questions and new insights.
        Science. 2011; 332: 1519-1523
        • Angulo P.
        Long-term mortality in nonalcoholic fatty liver disease: is liver histology of any prognostic significance?.
        Hepatology. 2010; 51: 373-375
        • Lu S.C.
        • Mato J.M.
        S-adenosylmethionine in liver health, injury, and cancer.
        Physiol Rev. 2012; 92: 1515-1542
        • Moylan C.A.
        • Pang H.
        • Dellinger A.
        • et al.
        Hepatic gene expression profiles differentiate presymptomatic patients with mild versus severe nonalcoholic fatty liver disease.
        Hepatology. 2014; 59: 471-482
        • Lu S.C.
        • Alvarez L.
        • Huang Z.Z.
        • et al.
        Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation.
        Proc Natl Acad Sci U S A. 2001; 98: 5560-5565
        • Barr J.
        • Caballeria J.
        • Martínez-Arranz I.
        • et al.
        Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression.
        J Proteome Res. 2012; 11: 2521-2532
        • Kleiner D.E.
        • Brunt E.M.
        • Van Natta M.
        • et al.
        Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
        Hepatology. 2005; 41: 1313-1321
        • Rousseeuw P.J.
        Silhouettes: a graphical aid to the interpretation and validation of cluster analysis.
        J Comput Appl Math. 1987; 20: 53-65
      1. Maechler M, Rousseuw PJ, Struyf A, et al. Cluster: cluster analysis basics and extensions. R package version 2.0.4. Available at https://www.r-project.org/.

        • Greco D.
        • Kotronen A.
        • Westerbacka J.
        • et al.
        Gene expression in human NAFLD.
        Am J Physiol Gastrointest Liver Physiol. 2008; 294: G1281-G1287
        • Vance D.E.
        Physiological roles of phosphatidylethanolamine N-methyltransferase.
        Biochim Biophys Acta. 2013; 1831: 626-632
        • Cano A.
        • Buque X.
        • Martinez-Una M.
        • et al.
        Methionine adenosyltransferase 1A gene deletion disrupts hepatic very low-density lipoprotein assembly in mice.
        Hepatology. 2011; 54: 1975-1986
        • Han M.S.
        • Park S.Y.
        • Shinzawa K.
        • et al.
        Lysophosphatidylcholine as a death effector in the lipoapoptosis of hepatocytes.
        J Lipid Res. 2008; 49: 84-97
        • Kakisaka K.
        • Cazanave S.C.
        • Fingas C.D.
        • et al.
        Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis.
        Am J Physiol Gastrointest Liver Physiol. 2012; 302: G77-G84
        • Loehrer F.M.
        • Schwab R.
        • Angst C.P.
        • et al.
        Influence of oral S-adenosylmethionine on plasma 5-methyltetrahydrofolate, S-adenosylhomocysteine, homocysteine and methionine in healthy humans.
        J Pharmacol Exp Ther. 1997; 282: 845-850
        • Mudd S.H.
        • Poole J.R.
        Labile methyl balances for normal humans on various dietary regimens.
        Metabolism. 1975; 24: 721-735
        • Lu S.C.
        • Ramani K.
        • Ou X.
        • et al.
        S-adenosylmethionine in the chemoprevention and treatment of hepatocellular carcinoma in a rat model.
        Hepatology. 2009; 50: 462-471
        • Murphy S.K.
        • Yang H.
        • Moylan C.A.
        • et al.
        Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease.
        Gastroenterology. 2013; 145: 1076-1087
        • Locasale J.W.
        Serine, glycine and one-carbon units: cancer metabolism in full circle.
        Nat Rev Cancer. 2013; 13: 572-583
        • Martinez-Chantar M.L.
        • Corrales F.J.
        • Martinez-Cruz L.A.
        • et al.
        Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A.
        FASEB J. 2002; 16: 1292-1294
        • Ji Y.
        • Nordgren K.K.
        • Chai Y.
        • et al.
        Human liver methionine cycle: MAT1A and GNMT gene resequencing, functional genomics, and hepatic genotype-phenotype correlation.
        Drug Metab Dispos. 2012; 40: 1984-1992
        • Kim J.-S.
        • Coon S.L.
        • Blackshaw S.
        • et al.
        Methionine adenosyltransferase:adrenergic-cAMP mechanism regulates a daily rhythm in pineal expression.
        J Biol Chem. 2005; 280: 677-684
        • Yang H.
        • Cho M.E.
        • Li T.W.
        • et al.
        MicroRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma.
        J Clin Invest. 2013; 123: 285-298
        • Anstee Q.M.
        • Day C.P.
        S-adenosylmethionine (SAMe) therapy in liver disease: a review of current evidence and clinical utility.
        J Hepatol. 2012; 57: 1097-1109

      References

        • Barr J.
        • Caballeria J.
        • Martinez-Arranz I.
        • et al.
        Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression.
        J Proteome Res. 2012; 11: 2521-2532
        • Martínez-Uña M.
        • Varela-Rey M.
        • Cano A.
        • et al.
        Excess S-adenosylmethionine reroutes phosphatidylethanolamine towards phosphatidylcholine and triglyceride synthesis.
        Hepatology. 2013; 58: 1296-1305
        • Martinez-Arranz I.
        • Mayo R.
        • Pérez-Cormenzana M.
        • et al.
        Enhancing metabolomics research through data mining.
        J Proteomics. 2015; 127: 275-288
        • van Liempd S.
        • Cabrera D.
        • Mato J.M.
        • et al.
        A fast method for the quantitation of key metabolites of the methionine pathway in liver tissue by high-resolution mass spectrometry and hydrophilic interaction ultra-performance liquid chromatography.
        Anal Bioanal Chem. 2013; 405: 5301-5310
        • Bligh E.G.
        • Dyer W.J.
        A rapid method of total lipid extraction and purification.
        Can J Biochem Physiol. 1959; 37: 911-917
        • Ruiz J.I.
        • Ochoa B.
        Quantification in the subnanomolar range of phospholipids and neutral lipids by monodimensional thin-layer chromatography and image analysis.
        J Lipid Res. 1997; 38: 1482-1489
        • Varela-Rey M.
        • Iruarrizaga-Lejarreta M.
        • Lozano J.J.
        • et al.
        S-adenosylmethionine levels regulate the Schwann cell DNA methylome.
        Neuron. 2014; 81: 1024-1039
        • Krueger F.
        • Andrews S.R.
        Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications.
        Bioinformatics. 2011; 27: 1571-1572
        • Akalin A.
        • Kormaksson M.
        • Li S.
        • et al.
        Methyl Kit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles.
        Genome Biol. 2012; 13: R87
        • Eng J.K.
        • Jahan T.A.
        • Hoopmann M.R.
        • et al.
        Comet: an open-source MS/MS sequence database search tool.
        Proteomics. 2013; 13: 22-24
        • Craig R.
        • Beavis R.C.
        TANDEM: matching proteins with mass spectra.
        Bioinformatics. 2004; 20: 1466-1467
        • Keller A.
        • Nesvizhskii A.I.
        • Kolker E.
        • et al.
        Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.
        Anal Chem. 2002; 74: 5383-5392
        • MacLean B.
        • Tomazela D.M.
        • Shulman N.
        • et al.
        Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.
        Bioinformatics. 2010; 26: 966-968
        • Schilling B.
        • Rardin M.J.
        • MacLean B.X.
        • et al.
        Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.
        Mol Cell Proteomics. 2012; 11: 202-214
        • Choi M.
        • Chang C.Y.
        • Clough T.
        • et al.
        MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments.
        Bioinformatics. 2014; 30: 2524-2526