Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles

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 biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; 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 VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG 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 LDL_5,6 , 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 patatin-like phospholipase domain-containing 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.

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), liver‐specific microsomal triglyceride transfer protein KO (Mttp‐LKO), liver‐specific transmembrane 6 superfamily member 2 KO (Tm6sf2‐LKO), and germline Ldlr‐deficient (Ldlr−/−.Leiden) KO mice fed high‐fat 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

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, intermediate‐density lipoprotein

FIGURE 3

VLDL‐TG and VLDL‐Apo‐B concentration by grade of steatosis for each NAFLD subtype and control cohort. (A,B) Total VLDL‐TG (A) and VLDL‐Apo‐B (B). Values are represented as means (points) and standard errors (vertical lines)

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)