Fig. 1: Identification and characterization of NASH-secreted proteins.

C57BL/6 mice were fed either a methionine- and choline-deficient diet (MCD, orange) or a diet enriched in lipid, fructose, and cholesterol (CHOL, green). Control mice received standard diet. a Hepatocytes from MCD and CHOL mice (and respective Controls) were isolated, followed by assessment of the intracellular proteome and secretome. b Liver weight (n = 4/group). *P = 0.049 MCD and *P = 0.011 CHOL Control vs. NASH, by unpaired two-tailed t test. c Representative liver histology (H&E and Masson’s Trichrome staining). Scale bar = 200 μm. Liver histology was assessed in three MCD Control, three MCD, four CHOL Control, four CHOL, with similar results obtained. d, e Hepatocyte mRNA expression of fibrosis and macrophage markers ((d) Control, n = 14/gene; MCD, n = 13/gene (d) MCD Pdgfb, n = 10 (e) Control, n = 9/gene (e) Control Pdgfb, n = 15 (e) CHOL), n = 10/gene). d *P = 0.002 Col1a1, *P = 0.008 Ctgf, *P = 0.005 Tgfb1, *P = 0.0002 Hsp47, P = 0.178 Pdgfb, *P = 0.002 Acta2, P = 0.054 F4/80 Control vs. NASH, by unpaired two-tailed t tests. e *P = 0.004 Col1a1, P = 0.184 Ctgf, *P = 0.002 Tgfb1, *P = 0.002 Hsp47, *P = 0.037 Pdgfb, P = 0.176 Acta2, *P = 0.003 F4/80 Control vs. NASH, by unpaired two-tailed t tests. f Volcano plots showing significant NASH-regulated secreted proteins from hepatocytes of MCD and CHOL-fed mice vs. Control mice. g Correlation of NASH-regulated changes in protein secretion from MCD and CHOL mice. h Ingenuity Pathway Analysis showing NASH-induced changes in diseases and functions. i–n Medium containing secreted factors derived from MCD (orange) and CHOL (green) hepatocytes was obtained, then applied to 3T3-L1 adipocytes, C2C12 myotubes and primary murine hepatocytes, followed by assessment of (i, j) basal and insulin-stimulated Akt S473 phosphorylation (n = 6/group), k 1-¹⁴C-2-deoxyglucose uptake in adipocytes (calculated as Δ, insulin-stimulated—basal) (n = 6/group for MCD, n = 5/group for CHOL), l adipocyte lipolysis (shown as Δ, isoproterenol-stimulated—basal) (n = 6/group, except MCD n = 5), m fatty acid oxidation in myotubes (n = 5/group), and n fatty acid uptake in hepatocytes (n = 6/group, except CHOL Control n = 5). C = control conditioned media; N = NASH conditioned media. For panel i, *P < 0.0001 Control and P = 0.064 MCD basal vs. insulin, adjoining line Control vs. NASH by two-way ANOVA and Bonferroni post hoc analysis (P = 0.027). For panel j, *P < 0.0001 Control and P = 0.548 CHOL Basal vs. Insulin, adjoining line Control vs. NASH by two-way ANOVA and Bonferroni post hoc analysis (P = 0.001). For panels k–n, *P < 0.05 by unpaired two-tailed t tests (k: *P = 0.041 MCD, *P = 0.042 CHOL; l: *P = 0.009 MCD, *P = 0.003 CHOL; m: *P < 0.0001 MCD, *P = 0.001 CHOL; N: *P = 0.009 CHOL, P = 0.178 MCD). For conditioned media experiments, each data point was obtained using secretion media from an individual mouse. o Classically secreted proteins significantly (P < 0.05, >twofold change) regulated by both MCD and CHOL dietary regimes. For panels d, e and i–n, data are means ± SEM. Source data are provided as a Source Data file. 2-DG 2-deoxyglucose, Acta2 smooth muscle alpha (α)-2 actin, Apoo apolipoprotein O, Arsa arylsulfatase A, Atp6ap1 ATPase H + transporting accessory protein 1, B Basal, C Control, CHOL diet enriched in lipid, sucrose, fructose and cholesterol, Col1a1 collagen type I alpha 1 chain, Cpn1 carboxypeptidase N subunit 1, Ctgf connective tissue growth factor, Ctsd cathepsin D, F4/80 adhesion G protein-coupled receptor E1 (ADGRE1), H&E hematoxylin and eosin, Hsp47 heat shock protein47, Hsd17b11 hydroxysteroid 17-beta dehydrogenase 11, I insulin, Lgals3 galectin 3, Lipc hepatic triacylglycerol lipase, MCD methionine–choline deficient, MT Masson’s Trichrome, N NASH, Ngp neutrophilic granule protein, Pdgfb platelet-derived growth factor, Pm20d1 peptidase M20 domain containing 1, Procr protein C receptor, Scpep1 serine carboxypeptidase 1, Tgfb1 transforming growth factor-beta, Ugt1a6 UDP glucuronosyltransferase 1 family member A6, Ugt2a3 UDP glucuronosyltransferase 2 family member A3, Ugt2b34 UDP glucuronosyltransferase 2 family member B34, Ugt2b36 UDP glucuronosyltransferase 2 family member B36.