Metabolomic Identification of Subtypes of Nonalcoholic Steatohepatitis - PubMed (original) (raw)

. 2017 May;152(6):1449-1461.e7.

doi: 10.1053/j.gastro.2017.01.015. Epub 2017 Jan 26.

David Fernández-Ramos 2, Marta Varela-Rey 2, Ibon Martínez-Arranz 1, Nicolás Navasa 2, Sebastiaan M Van Liempd 2, José L Lavín Trueba 2, Rebeca Mayo 1, Concetta P Ilisso 2, Virginia G de Juan 2, Marta Iruarrizaga-Lejarreta 1, Laura delaCruz-Villar 2, Itziar Mincholé 1, Aaron Robinson 3, Javier Crespo 4, Antonio Martín-Duce 5, Manuel Romero-Gómez 6, Holger Sann 7, Julian Platon 8, Jennifer Van Eyk 3, Patricia Aspichueta 9, Mazen Noureddin 10, Juan M Falcón-Pérez 2, Juan Anguita 2, Ana M Aransay 2, María Luz Martínez-Chantar 2, Shelly C Lu 10, José M Mato 11

Affiliations

Metabolomic Identification of Subtypes of Nonalcoholic Steatohepatitis

Cristina Alonso et al. Gastroenterology. 2017 May.

Abstract

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: 1-Carbon Metabolism; Lipid Metabolism; Mouse Model; Prognostic.

Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. SAMe depletion alters one-carbon metabolism

A) Schematic representation of one-carbon metabolism. One-carbon metabolism circulates one-carbon units from different inputs (methionine, choline, serine, threonine, glycine), via SAMe and methyltetrahydrofolate (MTHF), into a large variety of outputs, such as DNA and phospholipid methylation, glutathione (GSH), polyamines, NADPH and nucleotide synthesis. B) Relative fold-change (log2) in the hepatic content of the main metabolites involved in one-carbon metabolism in MAT1A-KO as compared to WT mice. MAT1A deletion induced a reduction in hepatic SAMe content and downstream metabolites, such as phosphatidylcholine with docosahexaenoic acid PC(22:6), methylthioadenosine (MTA, a biomarker of polyamine biosynthesis), GSH, hypotaurine (HTAU) and taurine (TAU) (three key metabolites of the transsulfuration pathway), NADPH and nucleotides. MAT1A ablation resulted also in the accumulation of methionine (Met) and upstream metabolites, such as serine (Ser), threonine (Thr), MTHF and phosphatidylethanolamine with docosahexaenoic acid PE(22:6). C) Relative fold-change (log2) in the protein content of enzymes involved in hepatic one-carbon metabolism in MAT1A-KO as compared to WT mice. MAT1A deletion led to abnormal protein content of numerous enzymes involved in one-carbon metabolism. AHCY, adenosylhomocysteinase; ALDH1A1, aldehyde dehydrogenase 1a1; BHMT, betaine-homocysteine S-methyltransferase; CBS, cystathionine β-synthase; CSAD, cysteine sulfinic acid decarboxylase; CTH, cystathionine γ-lyase; DMGDH, dimethylglycine dehydrogenase; MAT2A, methionine adenosyltransferase 2a; and SDS, serine dehydratase. *P<.05.

Figure 2

Figure 2. SAMe depletion activates FA uptake and esterification, while FA oxidation and VLDL secretion are impaired

A) Schematic representation of hepatic lipid metabolism. Hepatic fatty acids (FA) originate from serum and through de novo lipogenesis (DNL). FA can either be oxidized in the mitochondria (Mit) or esterified to form triglycerides (TG), which are stored in lipid droplets (LD), used to form other lipids, such as phospholipids, ceramides (Cer) and cholesteryl esters (ChoE) (not shown), or exported into blood as very low density lipoproteins (VLDL). The formation of VLDL particles requires phosphatidylcholine (PC) molecules rich in polyunsaturated FA (PUFA), such as PC(22:6). The rate-limiting step in mitochondrial β-oxidation is carnitine palmitoyltransferase 1a (CPT1A), which forms palmitoylcarnitine (AC16:0). The accumulation of FA in the cytoplasm increases their oxidation in peroxisomes (Px) and endoplasmic reticulum (ER). The first step in Px β-oxidation is acyl-CoA oxidase 1 (ACOX1), which generates reactive oxygen species (ROS). ER ω-oxidation, which is catalyzed by cytochrome P450 (CYP) enzymes, such as CYP2E1 and CYP4A10, also generates ROS. In its turn, ROS induces glutathione (GSH) depletion and produces oxidized FA (oxFA), such as linoleic acid (LA) derived oxidized FA (oxLA), which can lead to fibrosis and cell death. B) Relative fold-change (log2) in the hepatic content of the main metabolites involved in lipid metabolism in MAT1A-KO as compared to WT mice. AC(16:0), palmitoylcarnitine; oxFA, oxidized FA; oxLA, linoleic acid (18:2)-derived oxidized FA; PC, phosphatidylcholine; lyso-PC, lyso-phosphatidylcholine; PE, phosphatidylethanolamine; lyso-PE, lyso-phosphatidylethanolamine; PC(22:6)/PC, ratio PC with docosahexaenoic acid/total PC; PC(20:4)/PE(20:4) and ratio PC/PE with arachidonic acid. C) Relative fold-change (log2) in the content of proteins involved in liver lipid metabolism in MAT1A– KO as compared to WT mice. DNL enzymes: ACLY, citrate lyase; ACC1, acetyl-CoA carboxylase 1; FAS, and fatty acid synthase. FA transport: CD36, fatty acid translocase. FA esterification: SCD1, stearoyl-CoA desaturase; AGPAT2, 1-acylglycerol-3-phosphate O-acyltransferase 2; and DGAT2, diacylglycerol acyltransferase 2. Mitochondrial FA β-oxidation: CPT1A, carnitine palmitoyltransferase 1a; ACSM5, acyl-CoA synthetase medium chain family member 5; ACAD8; acyl-CoA dehydrogenase family member 8; and ALDH1B1; aldehyde dehydrogenase 1 family member B1.Peroxisomal FA β-oxidation: ACOX1, acyl-CoA oxidase 1; and ACAA1B, acyl-CoA acetyltransferase. Endoplasmic reticulum FA ω-oxidation: CYP2E1 and CYP4A10. *P<.05.

Figure 3

Figure 3. The serum metabolomic profile reflects hepatic metabolism

Comparison of liver and serum metabolomics profiles of MAT1A-KO mice. Each point represents the log2(fold-change) of individual metabolic ion features of MAT1A-KO compared to WT mice in serum and liver. A list with the log2(fold-change) and _P_-value for each individual metabolite in serum and liver is given in supplementary Table 1. R2=0.45, _P_=1E-04. AA, amino acids; AC, acyl carnitines; BA, bile acids; Cer, ceramides; CMH, monohexosylceramides; Cho, cholesterol; ChoE, cholesteryl esters; DG, diglycerides; FAA, fatty acyl amides; PC, phosphatidylcholines; Lyso-PC, lyso- phosphatidylcholines; PE, phosphatidylethanolamines; Lyso-PE, lyso-phosphatidylethanolamines; PI, phosphatidylinositols; Lyso-PI, lyso-phosphatidylinositols; MG, monoglycerides; SFA, MUFA and PUFA, saturated, monounsaturated and polyunsaturated fatty acids, respectively; NAE, N-acylethanolamines; SM, sphingomyelins; ST, steroids; TG, triglycerides.

Figure 4

Figure 4. Identification of a subset of NAFLD patients showing a _Mat1a_-KO serum metabolomic profile

A) Volcano plot representation indicating the -log10(_P_-value) and log2(fold-change) of individual serum metabolic ion features of MAT1A-KO compared to WT mice. AA, amino acids; AC, acyl carnitines; BA, bile acids; Cer, ceramides; CMH, monohexosylceramides; Cho, cholesterol; ChoE, cholesteryl esters; DG, diglycerides; FAA, fatty acyl amides; PC, phosphatidylcholines; Lyso-PC, lyso-phosphatidylcholines; PE, phosphatidylethanolamines; Lyso-PE, lyso-phosphatidylethanolamines; PI, phosphatidylinositols; Lyso-PI, lyso-phosphatidylinositols; MG, monoglycerides; SFA, MUFA and PUFA, saturated, monounsaturated and polyunsaturated fatty acids, respectively; NAE, N-acylethanolamines; SM, sphingomyelins; ST, steroids; TG, triglycerides. B) Heatmap representation of the serum metabolomic profile from 535 patients with biopsy-confirmed NAFLD. Each data point corresponds to the relative ion abundance of a given metabolite (vertical axis) in an individual patient’s serum. Metabolite selection is based on the top 50 serum metabolites that more significantly differentiated between MAT1A-KO and WT mice. The hierarchical clustering is based on optimum average silhouette width, obtaining the classification of the samples into two groups: first cluster resembles the serum metabolomic profile observed in the MAT1A-KO mice (M-subtype), while second cluster shows a different metabolomic profile (non-M-subtype).

Figure 5

Figure 5. Scheme for the identification and validation of NAFLD subtypes and NASH biomarkers

Serum samples from 535 patients with biopsy proven NAFLD (353 simple steatosis and 182 NASH) were randomly partitioned (50/50) into two cohorts (estimation and validation cohort) with equal proportion of steatosis/NASH and male/female. Clustering analysis, based in the 50 serum metabolites that more significantly differentiated between MAT1A-KO and WT mice (see Figure 4), generated two main clusters and patients were classified into M and non-M-subtypes. Based on the complete metabolic profile (N=328 metabolites) of the human serum samples, biomarkers that significantly differentiated between NASH and simple steatosis were selected and validated by comparison between the results in estimation and validation cohorts. After 1,000-fold repetition of this random partition, each time with equal proportional representation of simple steatosis/NASH and male/female, the frequency distribution of the metabolites that significantly differentiated between NASH and simple steatosis in the M- and non-M-subtypes was determined, and those showing a reproducibility of at least 700 times in 1,000 repetitions selected. The frequency distribution of the NAFLD patients into the M- and non-M-subtypes was also calculated. Following the criteria based on ≥70% reproducibility, 262 patients (49%) were classified as M-subtype and 171 (32%) as non-M-subtype. The remaining 102 patients (19%) showed a reproducibility of less than 70% and could not be classified as either M- or non-M-subtype (indeterminate group). PC, phosphatidylcholine; Lyso-PC, lyso- phosphatidylcholine; PE, phosphatidylethanolamine; Lyso-PE, lyso- phosphatidylethanolamine; PI, phosphatidylinositol.

Figure 6

Figure 6. Effect of SAMe administration on histology and serology in MAT1A KO mice

A) Representative images of hematoxylin and eosin (H&E), Sudan III red, Sirius red and F4/80 immunofluorescence staining of liver tissues after eight weeks SAMe (30 mg/kg/day) or vehicle administration are shown. Sizing bars correspond to 100 µm for H&E and Sirius Red, and 50 µm for Sudan III and F4/80. Quantitative analyses are shown in the table. Results that were significantly different (P<.05) to vehicle-given MAT1A-KO mice are indicated. Data shown represent mean of twelve vehicle-given MAT1A-KO, twelve SAMe-treated MAT1A-KO and eleven vehicle-given wild type (WT) animals. B) Effect of SAMe administration on serum parameters. For each group of animals (WT + vehicle, MAT1A-KO + vehicle, and MAT1A-KO + SAMe), results that were significantly different (P<.05) before and after administration are indicated. Twelve SAMe-given MAT1A-KO, twelve vehicle-treated MAT1A-KO and eleven vehicle-given WT mice were analyzed.

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