Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles

Hepatology. 2022; :1121–1134. | 1121 wileyonlinelibrary.com/journal/hep ORIGINAL ARTICLE Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles Ibon Martínez- Arranz1 | Chiara Bruzzone2 | Mazen Noureddin3 | Ruben Gil- Redondo2 | Itziar Mincholé1 | Maider Bizkarguenaga2 | Enara Arretxe1 | Marta Iruarrizaga- Lejarreta1 | David Fernández- Ramos2 | Fernando Lopitz- Otsoa2 | Rebeca Mayo1 | Nieves Embade2 | Elizabeth Newberry4 | Bettina Mittendorf5 | Laura Izquierdo- Sánchez6 | Vaclav Smid7 | Jorge Arnold8 | Paula Iruzubieta9 | Ylenia Pérez Castaño10 | Marcin Krawczyk11,12 | Urko M. Marigorta2 | Martine C. Morrison13 | Robert Kleemann13 | Antonio Martín- Duce14 | Liat Hayardeny15 | Libor Vitek7 | Radan Bruha7 | Rocío Aller de la Fuente16 | Javier Crespo9 | Manuel Romero- Gomez17 | Jesus M Banales6,18 | Marco Arrese8,19 | Kenneth Cusi20 | Elisabetta Bugianesi21 | Samuel Klein5 | Shelly C. Lu3 | Quentin M. Anstee22,23 | Oscar Millet2 | Nicholas O. Davidson4 | Cristina Alonso1 | José M. Mato2 1OWL Metabolomics, Derio, Bizkaia, Spain 2CIC bioGUNE, BRTA, CIBERehd, Derio, Bizkaia, Spain 3Karsh Division of Gastroenterology and Hepatology, Cedars- Sinai Medical Center, Los Angeles, California, USA 4Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA 5Center for Human Nutrition, Washington University School of Medicine, St. Louis, Missouri, USA 6Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, Donostia, Spain 7First Faculty of Medicine, Charles University, Prague, Czech Republic 8Departamento de Gastroenterologia, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago de Chile, Chile 9Marqués de Valdecilla University Hospital, Cantabria University, Santander, Spain 10Department of Digestive System, Osakidetza Basque Health Service, Donostia University Hospital, San Sebastian, Spain 11Department of Medicine II, Saarland University Medical Center, Homburg, Germany 12Laboratory of Metabolic Liver Diseases, Center for Preclinical Research, Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Warsaw, Poland 13Department of Metabolic Health Research, Netherlands Organization for Applied Scientific Research, Leiden, The Netherlands 14Alcalá University School of Medicine and Health Sciences, University Hospital Prıncipe de Asturias, Madrid, Spain 15Galmed Pharmaceuticals, Tel Aviv, Israel Received: 11 August 2021 | Revised: 18 January 2022 | Accepted: 15 February 2022 DOI: 10.1002/hep.32427 This is an open access article under the terms of the Creative Commons Attribution- NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2022 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases. Ibon Martínez- Arranz, Chiara Bruzzone, and Mazen Noureddin contributed equally to the study. Abbreviations: 0.1MCD, methionine (0.1%) and cholinedeficient diet; ALT, alanine aminotransferase; Apo, apolipoprotein; AST, aspartate aminotransferase; BMI, body mass index; CL, cholesterol; CoA, coenzyme A; CVD, cardiovascular disease; DG, diglycerides; DNL, de novo lipogenesis; FA, fatty acid; FCR, fractional catabolic rate; FRS, Framingham risk score; GGT, gammaglutamyltransferase; HbA1c, glycated hemoglobin; HFD, highfat diet; HOMA- IR, homeostasis model assessment of insulin resistance; IDL, intermediatedensity lipoprotein; IHTG, intrahepatic triglyceride; INR, international normalized ratio; KO, knockout; Ldlr, LDL receptor; Mat1a, methionine adenosyltransferase 1a; Mttp, microsomal triglyceride transfer protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PNPLA3, patatinlike phospholipase domaincontaining protein 3; RLP, remnant lipoprotein; SAMe, S- adenosylmethionine; SM, sphingomyelin; SR, secretion rate; TG, triglycerides; TM6SF2, transmembrane 6 superfamily 2. 76 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | METABOLIC SUBTYPES OF NAFLD INTRODUCTION NAFLD represents a spectrum of progressive liver disease characterized by increased intrahepatic triglyceride (IHTG) content in the absence of excessive alcohol consumption.[1– 3] NAFLD ranges from simple steatosis to NASH, fibrosis, and ultimately cirrhosis and HCC. NAFLD is strongly associated with obesity, dyslipidemia, type 2 diabetes mellitus, and the metabolic syndrome. Importantly, cardiovascular disease (CVD) is the leading cause of mortality in patients with NAFLD.[4] Although NAFLD has been shown to be an independent risk factor for CVD, the mechanisms by which NAFLD contributes to CVD are unknown. 16Department of Digestive Disease, Clinic University Hospital, University Hospital of Valladolid, Valladolid, Spain 17Valme University Hospital, CiBERehd, Sevilla, Spain 18University of the Basque Country, CIBERehd, IKERBASQUE, Donostia, Spain 19Centro de Envejecimiento y Regeneración, Santiago, Chile 20Division of Endocrinology, Diabetes and Metabolism, University of Florida and Malcom Randall VAMC, Gainesville, Florida, USA 21Gastroenterology Department, University of Turin, Turin, Italy 22Translational & Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK 23Newcastle NIHR Biomedical Research Center, Newcastle Upon Tyne Hospitals NHS Trust, Newcastle Upon Tyne, UK Correspondence Cristina Alonso, OWL Metabolomics, Parque Tecnológico de Bizkaia, 48160 Derio, Bizkaia, Spain. Email: [email protected] José M. Mato, CIC bioGUNE, Parque Tecnológico de Bizkaia, 48160 Derio, Spain. Email: [email protected] Funding information National Institutes of Health (R01DK123763, R01DK119437, HL151328, P30DK52574, P30DK56341, and UL1TR002345); Ministerio de Economía y Competitividad de España (SAF2017- 88041- R); Ministerio de Economía y Competitividad de España for the Severo Ochoa Excellence Accreditation (SEV- 2016- 0644); CIBERehd (Biomedical Research Center in Hepatic and Digestive Diseases) and Netherlands Organization for Applied Scientific Research Program (PMC13 and PMC15); Spanish Carlos III Health Institute (PI15/01132 and PI18/01075); Miguel Servet Program (CON14/00129 and CPII19/00008); Fondo Europeo de Desarrollo Regional, CIBERehd, Department of Industry of the Basque Country (Elkartek: KK- 2020/00008); La Caixa Scientific Foundation (HR17- 00601); Liver Investigation: Testing Marker Utility in Steatohepatitis consortium funded by the Innovative Medicines Initiative Program of the European Union (777377), which receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA; Newcastle NIHR Biomedical Research Center; Czech Ministry of Health (RVOVFN64165/2020); Fondo Nacional De Ciencia y Tecnología de Chile (1191145); and the Comisión Nacional de Investigación, Ciencia y Tecnología (AFB170005, CARE Chile UC); Agencia Nacional de Investigación y Desarrollo (ANID ACE 210009); European Union's Horizon 2020 Research and Innovation Program (825510). Abstract Background and Aims: We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors. Approach and Results: We analyzed serum metabolome from 1154 individuals with biopsyproven NAFLD, and from four mouse models of NAFLD with impaired VLDL- triglyceride (TG) secretion, and one with normal VLDLTG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDLTG secretion; subtype C phenocopied the metabolome of mice with normal VLDLTG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDLTG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDLTG and VLDL– apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6, and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10- year high risk of CVD, measured with the Framingham risk score, and the frequency of patatinlike phospholipase domaincontaining protein 3 NAFLD risk allele were lower in subtype A. Conclusions: Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification. 1122 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | HEPATOLOGY NAFLD represents a complex heterogeneous disease in which the histological features result from an imbalance in liver lipid metabolism caused by multiple nutritional and environmental exposures on a susceptible genetic background.[3,5– 7] A better understanding of NAFLD heterogeneity and its biology will facilitate the development of personalized treatments. Overproduction of VLDL into circulation has been observed in patients with NAFLD and is thought to be, in part, a consequence of increased de novo lipogenesis (DNL) promoting hepatic triglyceride (TG) synthesis.[8– 10] NAFLD is characterized by increased serum levels of TG and cholesterol (CL) in VLDL and intermediatedensity lipoproteins (IDL), increased concentration of smaller LDL subclasses, and decreased content of larger LDL subclasses, all of which are features associated with increased CVD risk.[11,12] Moreover, it has been suggested that increased levels of total lipids and CL in VLDL and LDL subclasses may be associated with NAFLD progression.[13] On the other hand, VLDLTG secretion is relatively impaired in a subset of patients with NAFLD and cannot compensate for increases in both fatty acid (FA) influx and DNL.[14] Genetic defects (e.g., microsomal triglyceride transfer protein [MTTP], transmembrane 6 superfamily member 2 [TM6SF2] and apolipoprotein B, [APO- B]), which impair hepatic VLDL secretion, are also associated with reduced LDLCL levels and a favorable CVD risk profile, yet they still cause steatosis that may progress to NASH with fibrosis, even without obesity or insulin resistance.[15– 20] In addition, the therapeutic benefits of LDL reduction in patients treated with antisense APO- B or MTTP inhibitors are associated with hepatic steatosis, inflammation and fibrosis, which limits their utility.[21– 23] Our published data, using highthroughput metabolomics, suggested that patients with NAFLD could be subclassified into two main metabolic subtypes.[24] One of these subtypes (which we here refer to as subtype A) corresponds to specific serum metabolic alterations that correlated with those observed in methionine adenosyltransferase 1A knockout (Mat1a- KO) mice,[24] a model of NASH characterized by a reduction in hepatic S- adenosylmethionine (SAMe),[25] impaired phosphatidylcholine (PC) synthesis, and VLDL secretion.[26] Importantly, patients with NAFLD often exhibit downregulation of MAT1A expression[27,28] and low SAMe.[29] The comparison of the serum lipidomic profile of LDL receptor (Ldlr)– KO mice fed a highfat diet[30] with the lipidomic profile of the cohort of patients with NAFLD mentioned previously revealed the existence of a second NAFLD subtype, referred to here as subtype C. The deletion of Ldlr in mice results in increased serum level of VLDLTG and LDLCL.[31– 33] These findings raised the corollary question of whether patients with NAFLD with subtype A show impaired secretion rate (SR) of VLDL compared to patients with NAFLD with subtype C, which would show VLDL hypersecretion, and, consequently, both NAFLD subtypes could present different CVD risks. Based on these concepts, here we investigated the association between NAFLD subtypes with the main classes (VLDL, IDL, LDL, and HDL) and subclasses of serum lipoproteins, as well as with several common singlenucleotide polymorphisms associated with hepatocyte TG accumulation and VLDL secretion. MATERIALS AND METHODS Study participants NAFLD cases Patients with biopsied NAFLD (n = 1154) were recruited at several clinical centers from Europe (Spain, UK, Italy, the Czech Republic), the USA (California and Florida), and Chile, as well as patients recruited by Galmed Pharmaceuticals (Israel) (Table 1). Patients fulfilled the following inclusion criteria: (1) age 18−75 years; (2) no known acute or chronic disease except for obesity, dyslipidemia, hypertension, or type 2 diabetes based on medical history, physical examination, and standard laboratory tests; and (3) alcohol consumption lower than 20 g/day for women and 30 g/day for men. Exclusion criteria included patients with NAFLD with cirrhosis and other causes of liver disease. The institutional review board of the participating hospitals approved the study, and written informed consent was obtained from all patients.[34] Diagnoses were established histologically in liver biopsy specimens. The histological diagnosis of NAFLD was established by a single liver pathologist in each hospital using the scoring system defined by Kleiner/ Brunt.[35,36] Following assessment, patients were classified into two histological groups: (1) biopsies with only steatosis, steatosis with inflammation but without ballooning, or steatosis with ballooning but without inflammation were classified as nonalcoholic fatty liver, and (2) the histopathologic definition of NASH was determined by the joint presence of steatosis, lobular inflammation, and hepatocellular ballooning, independently of the total NAFLD activity score. Blood was drawn under fasting conditions within 30 days of the diagnostic liver biopsy was performed. Serum was stored at −80°C until analysis as described.[34] Clinical data were collected retrospectively using patient records and laboratory values obtained at the time of biopsy. Serum samples from all 1154 patients with NAFLD were used to identify the main metabolic NAFLD subtypes by analyzing their serum metabolomic profiles. A subset of 197 patients with NAFLD from this cohort was used to study the association of the NAFLD metabolic subtype with the serum lipoprotein profile (Table S1). This cohort was determined by the availability of sufficient serum to perform the lipoprotein profiling (0.5 ml). For details see the Supporting Methods. 1123 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License METABOLIC SUBTYPES OF NAFLD Controls As a control for comparison of the lipoprotein profiling data, we included 350 serum samples from the general population[37] with similar gender and age to the cohort of patients with NAFLD used to determine the serum lipoprotein profile, and normal glucose, TG, CL, and alanine aminotransferase (ALT) (Table S2). For details, see the Supporting Methods. Subjects with plasma VLDLTG secretion rate Serum samples from women (n = 30) for which the hepatic VLDLTG metabolism and kinetics were previously studied[38] were used to determine the association of the NAFLD metabolic subtypes with the VLDLTG secretion rate (SR). Three groups were considered of the following characteristics: lean subjects with low VLDLTG SR (n = 10), obese subjects with NAFLD with TABLE 1 Clinical and biochemical characteristics of the patients with NAFLD included in the lipidomic study Total Subtype A Subtype B Subtype C p value Demographics n1154 541 305 308 Age (years) 50.0 ± 11.9 (1154) 47.9 ± 12.2 (541) 52.0 ± 11.3 (305) 51.7 ± 11.3 (308) 0.182 Female (%) 576 (50%) 266 (49%) 151 (49%) 159 (52%) 0.778 BMI (kg/m2) 34.6 ± 7.7 (1154) 36.4 ± 9.0 (541) 32.7 ± 6.0 (305) 33.4 ± 5.9 (308) 0.028 Biochemistry TG (mg/dl) 165.4 ± 106.1 (1109) 140.6 ± 94.5 (525) 177.5 ± 86.7 (292) 197.6 ± 130.2 (292) 2.84E- 17 Cholesterol (mg/dl) 189.3 ± 44.4 (862) 175.8 ± 41.1 (392) 188.0 ± 37.3 (226) 212.3 ± 46.4 (244) 2.86E- 16 ALT (U/l) 56.6 ± 44.9 (1112) 53.1 ± 45.5 (509) 56.3 ± 41.4 (297) 62.6 ± 46.5 (306) 6.20E- 04 AST (U/l) 38.7 ± 28.2 (1113) 35.2 ± 25.1 (509) 40.3 ± 29.3 (297) 43.1 ± 31.3 (307) 8.00E- 06 GGT (U/l) 66.5 ± 95.6 (687) 60.3 ± 104.5 (328) 70.5 ± 88.0 (190) 73.9 ± 85.0 (169) 2.09E- 04 HbA1c (%) 6.5 ± 1.2 (722) 6.5 ± 1.3 (259) 6.4 ± 1.0 (220) 6.5 ± 1.1 (243) 0.854 HOMA- IR 7.0 ± 7.7 (597) 6.7 ± 7.9 (204) 7.1 ± 6.5 (186) 7.3 ± 8.5 (207) 0.412 Albumin (g/dl) 4.4 ± 0.4 (897) 4.4 ± 0.4 (379) 4.5 ± 0.3 (261) 4.4 ± 0.4 (257) 3.49E- 04 INR 1.0 ± 0.2 (419) 1.1 ± 0.1 (138) 1.1 ± 0.3 (146) 1.0 ± 0.1 (135) 5.00E- 06 Medication Lipidlowering medication N685 (79.4%) 331 (76.3%) 193 (82.5%) 161 (82.6%) 0.076 Y178 (20.6%) 103 (23.7%) 41 (17.5%) 34 (17.4%) CVD risk (FRS) Intermediate– high (>10%) 46 (12.8%) 28 (12.1%) 8 (13.1%) 10 (14.7%) 0.851 High (>15%) 21 (5.8%) 10 (4.3%) 6 (9.8%) 5 (7.4%) 0.221 Histology NASH (%) 735 (64%) 359 (66%) 187 (61%) 189 (61%) 0.208 Steatosis grade (%) 148.31% 44.92% 52.13% 46.75% 0.118 231.81% 34.01% 30.82% 29.22% 319.88% 21.07% 17.05% 24.03% Inflammation grade (%) 012.39% 13.31% 12.13% 11.36% 0.059 146.94% 50.65% 46.56% 41.88% 233.97% 29.57% 34.75% 41.23% 3 6.68% 6.47% 6.56% 5.52% Ballooning grade (%) 029.54% 25.88% 35.08% 35.71% 0.013 145.73% 49.17% 42.30% 43.83% 224.71% 24.95% 22.62% 20.45% Fibrosis stage (%) 0 25.92% 26.80% 29.51% 28.25% 0.447 135.66% 38.26% 37.70% 37.34% 219.32% 22.74% 18.36% 18.18% 312.88% 12.20% 14.43% 16.23% Note: Values for age, BMI, HbA1c, TG, ALT, AST, GGT, HbA1c, HOMA- IR, albumin, and INR are given as means ± SD. Statistical significance was determined by Chisquare test for categorical variables and Kruskal- Wallis H- test for continuous variables. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CVD, cardiovascular disease; FRS, Framingham risk score; GGT, gammaglutamyl transferase; HbA1c, glycated hemoglobin; HOMA- IR, homeostasis assessment model for insulin resistance; INR, international normalized ratio. | 1124 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HEPATOLOGY low VLDLTG SR (n = 10), and obese subjects with NAFLD with high VLDLTG SR (n = 10). Animals Serum samples from five mouse models of NASH, all of them previously described, were used to compare their metabolomic profile with that of the patients with NAFLD mentioned previously: Mat1a- KO mice,[24] methionine (0.1%) and cholinedeficient diet (0.1MCD)– fed mice,[39] Mttp- LKO mice,[40] Tm6sf2- LKO mice,[41] and LDLRKO mice fed a highfat diet (Ldlr−/−.Leiden/ HFD).[30] Metabolomics Serum metabolomic profiles were semiquantified by liquid chromatography coupled with mass spectrometry.[24] For details see the Supporting Methods. Lipoprotein analyses Nuclear magnetic resonance lipoprotein analyses were performed in a subcohort of patients with biopsied NAFLD (n = 197; Supporting Table S1) and controls (n = 350; Supporting Table S2) as described.[37] For details, see the Supporting Methods. Statistical analysis Continuous variables were described by means ± SDs. Comparisons between pairs of groups were performed by twotailed independent Student t test or Kruskal- Wallis H- test. Chisquare test was applied for categorical variables. Unadjusted p values were considered as statistically significant below 0.05. Hierarchical clustering algorithms based on metabolites were used to visualize the differences in signatures between samples, considering the Ward’s minimum variance as the agglomeration method. The maximum of the average of the individual silhouette widths was calculated for the clusters. A multinomial logistic regression classifier analysis was applied to generate a predictive metabolic signature capable of discriminating among NAFLD subtypes. A forward stepwise method was used as a variable selection criterion, in which the process started with a null model and optimum variables were added in each step until the best model was achieved. Logarithmic transformation of the data was applied. Validation process was performed through 5- fold cross validation. References for libraries are included in the Supporting Methods. RESULTS The serum lipidome of patients with NAFLD with subtype A is similar to that of mouse models of NAFLD exhibiting defective VLDL secretion Comparison of the serum metabolomic profile of the patients with NAFLD (n = 1154; Table 1) with that of different mouse models of NASH showing impaired (Mat1a- KO, 0.1MCD, Mttp- KO, and Tm6sf2- KO) or increased (Ldlr−/−.Leiden/HFD) VLDLTG secretion using the experimental procedure previously described[24] resulted in the identification of three major metabolic NAFLD subtypes (A, B, and C) (Figure 1A, Figure S1). Patients with NAFLD (541, 47%) reflecting metabolomic features common to the four mouse models of NASH showing impaired VLDL secretion were classified as subtype A. Of the remaining subjects, 308 (27%) patients with NAFLD resembled the metabolic profile of the Ldlr−/−.Leiden/HFD NASH mouse model and were classified as subtype C, and the remaining 305 (26%) patients were classified as subtype B (Figure 1B, Table 1). We then searched for lipid species that best differentiated among A, B, and C NAFLD subtypes (Figure 1C), and generated a statistical model in which these given lipids were assigned a relative weight depending on their importance for the classification of patients with NAFLD into subtypes (Table S3). This set of 12 NAFLD subtype biomarkers was genderindependent. We also identified signatures that could differentiate steatosis from NASH for each subtype. These signatures included glycerolipids, glycerophospholipids, and sphingolipids (Figure S2). Table 1 lists the main clinical data of patients with NAFLD classified by their metabolic subtype. No differences were found between the percentage of patients with NASH or stage of fibrosis and the NAFLD subtype. Moreover, no gender differences were indicated between subtypes. Patients with metabolic subtypes B and C were slightly older, with lower body mass index (BMI), and had more frequently higher TG, CL, ALT, aspartate aminotransferase (AST), and gammaglutamyltransferase (GGT), and less ballooning than subjects with subtype A. The homeostasis model assessment of insulin resistance (HOMA- IR) index, glycated hemoglobin (HbA1c), albumin, and international normalized ratio (INR) were similar among the three subtypes. The Framingham CVD risk score (FRS) was calculated for 360 patients with NAFLD, who were chosen based on availability of clinical data. The percent of patients at intermediate CVD risk (FRS ≥ 10% at 10 years) was similar in subtypes A (12%), B (13%), and C (15%). When the percentage of patients at high CVD risk (FRS ≥ 15% at 10 years) was compared, patients with subtype A showed a lower score (4%) than patients with subtypes B (10%) or C (7%). We also found that the percent of patients with NAFLD | 1125 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License METABOLIC SUBTYPES OF NAFLD | 1126 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HEPATOLOGY treated with lipidlowering medication (statins and/or fibrates) was higher in subtype A (24%) than in subtypes B (17%) and C (17%), although it did not reach statistical significance (Table 1). These results indicate that lipidlowering medication has limited relevance to a particular NAFLD subtype. Serum VLDLTG concentration is lower among patients with NAFLD with subtype A compared with the subtype C Because the lipidomic profile of patients with NAFLD with subtype A resembled that of mouse models of NAFLD deficient in VLDLTG secretion, we hypothesized that the serum concentration of VLDLTG and its subclasses would also be lower in patients with NAFLD with subtype A as compared to patients with NAFLD with subtype C, and that patients with NAFLD with subtype B would show an intermediate content of total VLDLTG and subclasses. Comparison of the serum lipoprotein profile of a subset of 197 samples from the NAFLD cohort (Table S4) confirmed this hypothesis. Table S1 lists the main clinical data of this subset of patients with NAFLD classified by their metabolic subtype. As expected, our results indicate that, independently of their subtype, patients with NAFLD have higher serum TG levels (p < 0.0001) than controls (Figure 2, Table S4). Our data also show that the concentration of serum TG was higher (p < 0.0001) among patients with NAFLD with subtype C than among subjects with NAFLD with subtype A, with intermediate levels of TG in patients with NAFLD with subtype B (Figure 2, Table S4). In accordance with former studies in patients with NAFLD,[10,13] we observed that, independently of the subtype, the concentration of TG in VLDL was higher in the patients with NAFLD (p < 0.0001) compared with controls (Figure 2, Table S4). Furthermore, our results show that the concentration of TG in VLDL, IDL, and LDL was lower (p < 0.0001) in patients with subtype A than among patients with subtype C, with intermediate levels of VLDLTG, IDLTG, and LDLTG in patients with NAFLD with subtype B (Figure 2). The concentration of serum VLDL- Apo- B (a surrogate for VLDL particle number) was higher among patients with NAFLD with subtype C than among subjects with subtype A (p < 0.0001), with intermediate levels of VLDL- Apo- B in patients with subtype B (Figure 2). The difference in serum IDL- Apo- B concentration between the A and C subtypes, although statistically significant (p < 0.0001), was less pronounced than for VLDL- Apo- B (Figure 2). For LDL- Apo- B, patients with subtype C showed higher levels than patients with subtype A (p < 0.0001), with intermediate levels in patients with subtype B (Figure 2). The concentration of LDL- Apo- B was higher among the control group than among patients with subtype A (p < 0.0001), and lower than in the cases with subtype C (Figure 2). This observation is reflected when the total concentration of Apo- B was determined, which follows the order: subtype A < control group < subtype C (Figure 2). Given that a reduction in VLDLTG in patients with NAFLD with subtype A might reflect partitioning of acetyl– coenzyme A (CoA) toward ketone body synthesis, we determined the serum concentration of acetoacetate, βhydroxybutyrate, and acetone in the control group and the NAFLD subtypes. Our results showed that there are no differences in the concentration of ketone bodies among subtypes (Figure S3). In patients with NAFLD with subtype A, the serum concentration of VLDLTG and VLDL- Apo- B was independent of the degree of steatosis We next examined the relationship between serum VLDLTG and VLDL- Apo- B concentrations and the grade of steatosis for each NAFLD subtype. Liver steatosis was graded histologically based on the percentage FIGURE 1 Classification of patients with NAFLD (n = 1242) into subtypes. (A) The frequency distribution of the patients with NAFLD (n = 1154) according to five mouse models of NAFLD. Frequencies were obtained after (1) random patient partition (50/50) into two cohorts with equal proportional representation of nonalcoholic fatty liver (NAFL)/NASH and males/females; (2) clustering analysis based on the 50 more significantly serum metabolites that differentiated more significantly between the mouse model of NAFLD and the respective control mice; (3) 1000- fold repetition of the random partition with equal representation of NAFL/NASH and males/females. Representation of the number of repeats associated with methionine adenosyltransferase 1A knockout (Mat1a- KO) mice, C57BL/6 mice fed 0.1% methionine and choline deficient diet (0.1MCD), liverspecific microsomal triglyceride transfer protein KO (Mttp- LKO), liverspecific transmembrane 6 superfamily member 2 KO (Tm6sf2- LKO), and germline Ldlrdeficient (Ldlr−/−.Leiden) KO mice fed highfat diet (HFD). Patients with a frequency over 70% for Mat1a- KO, 0.1MCD, Mttp- LKO and Tm6sf2- LKO, and lower than 70% for Ldlr−/−.Leiden/HFD, were classified as subtype A (colored in red). Patients with a frequency lower than 70% for Mat1a- KO, 0.1MCD, Mttp- LKO and Tms6f2- LKO, and higher than 70% for the Ldlr−/−.Leiden/HFD, were classified as subtype C (colored in blue). Patients not classified as either subtype A or C were classified as subtype B (colored in yellow). (B) Percentage of patients classified as subtypes A, B, and C. Percentage of patients with NAFL and NASH per subtype is also indicated. (C) Serum levels of lipids that discriminate among NAFLD subtypes A, B, and C. Gray points in the background indicate the real values with a minimal random displacement to avoid overplotting. Densities are represented as violin plots, while main distributions (medians and first and third quartiles) are represented as internal box plots. Horizontal black lines are statistical comparisons (t test) between two groups, with the unadjusted p values as symbols above the lines (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, ≥ 0.05). ns, not significant; PC, phosphatidylcholine; SM, sphingomyelin; TG, triglycerides | 1127 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 28 METABOLIC SUBTYPES OF NAFLD of hepatocytes showing fat accumulation[36]: S0 (normal, <5%), S1 (mild, 5%– 33%), S2 (moderate, 34%– 66%), and S3 (severe, >66%). When the serum concentrations of VLDLTG and VLDL- Apo- B from patients with NAFLD (n = 197) and the control group (n = 350) were plotted against the grade of steatosis for each NAFLD subtype, we found that patients with subtype C showed a curvilinear relationship between these parameters with a peak at S2, whereas in patients with subtype B increased linearly from S1 to S3 (Figure 3). In contrast, patients with subtype A exhibited lower concentrations of serum VLDLTG and VLDL- Apo- B, regardless of the steatosis grade (Figure 3). Serum albumin and INR were comparable among subtypes (Table 1), indicating that altered hepatic VLDL production alone does not account for the differences in hepatic steatosis in subtype A. Cardiovascular risk factors are lower among patients with NAFLD with subtype A compared with subtype C Compared to the control group, patients with NAFLD with subtype C exhibited an increase in the serum concentration of VLDL and small dense LDL (LDL5 and LDL6) particles, lower total HDLCL, higher LDLCL/ HDLCL and Apo- B/Apo- A1 ratios, and increased concentration of remnant lipoprotein cholesterol (RLPCL, the sum of the CL associated with VLDL and IDL) (Figure 4, Table S4), which are all markers of CVD risk and have previously been associated with NAFLD.[4] Our data also show that the concentration of VLDL and small dense LDL (LDL5 and LDL6) particles, the HDLCL content, and the RLPCL concentration were lower among patients with subtype A than among subjects with subtype C, whereas the LDLCL/HDLCL ratio showed similar values in both subtypes (Figure 4). Patients with B subtype exhibited intermediate values for these parameters. These results indicate that, compared to the C subtype, patients with NAFLD with subtype A exhibited a favorable CVD risk profile. VLDLTG SR is lower in subjects with NAFLD with subtype A compared to the B and C subtypes To address the question of whether patients with NAFLD with subtype A have lower VLDLTG SR than patients with non- A subtype, we examined the FIGURE 2 Blood lipoprotein TG (A– D) and apolipoprotein B (Apo- B) (E– H) concentrations for NAFLD subtypes and control subjects. Gray points in the background indicate the real values with a minimal random displacement to avoid overplotting. Densities are represented as violin plots, while main distributions (medians and first and third quartiles) are represented as internal box plots. Horizontal black lines are statistical comparisons (t test) between two groups, with the unadjusted p values as symbols above the lines (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, ≥ 0.05). IDL, intermediatedensity lipoprotein (A) (B) (C) (D) (E) (F) (G) (H) FIGURE 3 VLDLTG and VLDL- Apo- B concentration by grade of steatosis for each NAFLD subtype and control cohort. (A,B) Total VLDLTG (A) and VLDL- Apo- B (B). Values are represented as means (points) and standard errors (vertical lines) (A) (B) | 11 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License HEPATOLOGY relationship between the NAFLD subtype, the plasma VLDLTG concentration, and the VLDLTG SR in a group of obese women (n = 20) with IHTG content assessed by MR spectroscopy. A group of lean women (n = 10) with low VLDLTG SR and normal IHTG content was included for comparison (Table 2). These subjects are a subgroup of a larger cohort previously used to study VLDLTG SR kinetics.[38] Subtyping of the obese subjects with NAFLD was performed using the classification algorithm described in Table S2. Of the 20 obese subjects with NAFLD studied, 12 fell into subtype A, 5 into subtype B, and 3 into subtype C (Table 2). Subjects with subtypes B and C were grouped together for comparative reasons. Obese subjects with NAFLD with subtype A had similar age, BMI, and IHTG content than patients with subtypes B and C. As hypothesized, VLDLTG SR was higher (2.7- fold) among obese subjects with NAFLD with subtypes B and C than in obese subjects with NAFLD with subtype A (Table 2). The fractional catabolic rate (FCR, the fraction of the intravascular pool of VLDL catabolized per hour) was similar among obese subjects with NAFLD with subtype A that in the patients with subtypes B and C (Table 2). Obese patients with NAFLD with subtype A had similar plasma TG, VLDLTG, and VLDLTG SR than lean subjects (Table 2). Relevance of the genotype to a particular NAFLD subtype To study the relevance of the genotype to a particular NAFLD metabolic phenotype, we tested the association with the three NAFLD subtypes for four of the major known genetic risk factors, namely, rs9992651 (17β- hydroxysteroid dehydrogenase type 13), rs641738 (membranebound O- acyltransferase domaincontaining protein 7), rs738409 (patatinlike phospholipase domaincontaining protein 3 [PNPLA3]), and rs58542926 (TM6SF2).[5] Only one variant (PNPLA3) associated with metabolic subtype distributions (p = 0.0003) (Table 3), being that this allele is overrepresented in individuals with NAFLD subtypes B and C. The I148M protein variant of PNPLA3 accounts for the largest proportion of genetic predisposition to NAFLD in all populations.[42– 44] This variant has been associated with increased hepatic fat and elevated liver enzymes, which goes in accordance with the biological FIGURE 4 Blood lipoprotein particle numbers and cholesterol concentrations for NAFLD subtypes (A, B, and C) and control subjects. (A) VLDL particle number. (B) LDL- 5 particle number. (C) LDL- 6 particle number. (D) HDL cholesterol. (E) LDL cholesterol/HDL cholesterol ratio. (F) Remnant lipoprotein cholesterol. Gray points in the background indicate the real values with a minimal random displacement to avoid overplotting. Densities are represented as violin plots, while main distributions (medians and first and third quartiles) are represented as internal box plots. Horizontal black lines are statistical comparisons (t test) between two groups, with the unadjusted p values as symbols above the lines (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, ≥ 0.05) (A) (B) (C) (D) (E) (F) | 1129 15273350, 2022, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32427 by Universidad De Sevilla, Wiley Online Library on [01/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License