Journal: Hepatology Communications

Article Title: B4GALT1 deficiency attenuates steatohepatitis by regulating the PPARγ/ACSL4 axis

doi: 10.1097/HC9.0000000000000920

Figure Lengend Snippet: B4GALT1 is upregulated in patients with MASLD and a murine model of MASLD. (A) Relative B4GALT1 mRNA levels of non-steatosis (n=2), low steatosis (n=6), and high steatosis (n=10) in GSE46300 and of NAS 1–2 (n=12), NAS 3–4 (n=26), NAS≥5 (n=56), F0–F1 (n=36), and F2–F4 (n=58) in GSE174478 datasets. Box and whisker plots display the median (horizontal bar), interquartile range (box limits), and 1.5×interquartile range (whiskers). (B) Representative immunohistochemistry (IHC) images and quantified staining intensity of B4GALT1 classified according to NAS and fibrosis stage in patients with MASLD (NAS 1–2, n=5; NAS 3–4, n=6; NAS≥5, n=19; F0–F1, n=14; F2–F4, n=16). Scale bars, 50 µm. (C) Correlation analysis of serum ALT, TG, and CAP score with B4GALT1 in human MASLD liver samples (n=30). (D) mRNA and (E) protein levels of B4GALT1 in the livers of C57BL/6 mice fed a chow diet or CDAHFD. (F) Representative IHC images of B4GALT1 and RCA-I in liver samples from the control and CDAHFD-fed mice. Scale bars, 100 µm. (G) Quantification of the staining intensity of B4GALT1 and RCA-I in (F) (n=6/group). (H) B4GALT1 and RCA-I protein levels in AML12 cells treated with 0.5% BSA or 0.5 mM FFA. Data are presented as mean ± SEM in (B), (D), and (G). For (A), (B), (D), and (G), significance was determined by the Student t test or the ANOVA (* p <0.05, ** p <0.01, **** p <0.0001). Abbreviations: B4GALT1, β-1,4-galactosyltransferase 1; BSA, bovine serum albumin; CDAHFD, choline-deficient, L–amino acid–defined, high-fat diet; FFA, free fatty acids; MASLD, metabolic dysfunction–associated steatotic liver disease; NAS, NAFLD activity score; RCA-I, Ricinus communis agglutinin I.

Article Snippet: Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and non-esterified fatty acids (NEFA) as well as the TG, TC, LDL, and NEFA levels in murine liver tissues were quantified following standardized manufacturer protocols (ALT: cat. no. C009-2; AST: cat. no. C010-2; TG: cat. no. A110-1; TC: cat. no. A111-1; LDL: cat. no. A113-1; NEFA: cat. no. A042-2; Nanjing Jiancheng).

Techniques: Whisker Assay, Immunohistochemistry, Staining, Control, Activity Assay

Journal: Hepatology Communications

Article Title: B4GALT1 deficiency attenuates steatohepatitis by regulating the PPARγ/ACSL4 axis

doi: 10.1097/HC9.0000000000000920

Figure Lengend Snippet: Hepatic loss of B4galt1 alleviates fatty degeneration and inflammation in MASLD mice. (A) Schematic diagram of a murine model fed a chow diet or CDAHFD. (B) Body weight (n=8–10/group) and liver weight/body weight (%) (n=6–8/group) were measured in B4galt1 flox/flox and B4galt1 hep−/− mice fed a chow diet or CDAHFD. (C) Serum levels of ALT and AST were measured (n=6–8/group). (D) Levels of TNF-α and IL-6 in the liver tissues were detected using ELISA (n=6–8/group). (E) Serum and hepatic levels of TG, TC, LDL, and NEFA were tested (n=5–8/group). (F) Sections of paraffin-embedded liver tissue were subjected to H&E, Sirius red staining, and immunohistochemical staining for α-SMA and F4/80. Frozen sections were stained with Oil Red O. Scale bars, 100 µm. (G) Steatosis, lobular inflammation scores, and total NAS for each group (n=8–10/group). (H) Quantification of Oil Red O, F4/80, and Sirius red staining. Data are presented as mean ± SEM (* p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001; ANOVA). Abbreviations: B4GALT1, β-1,4-galactosyltransferase 1; CDAHFD, choline-deficient, L–amino acid–defined, high-fat diet; ELISA, enzyme-linked immunosorbent assay; H&E, hematoxylin and eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; NAS, NAFLD activity score.

Article Snippet: Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and non-esterified fatty acids (NEFA) as well as the TG, TC, LDL, and NEFA levels in murine liver tissues were quantified following standardized manufacturer protocols (ALT: cat. no. C009-2; AST: cat. no. C010-2; TG: cat. no. A110-1; TC: cat. no. A111-1; LDL: cat. no. A113-1; NEFA: cat. no. A042-2; Nanjing Jiancheng).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, Activity Assay

Journal: Hepatology Communications

Article Title: B4GALT1 deficiency attenuates steatohepatitis by regulating the PPARγ/ACSL4 axis

doi: 10.1097/HC9.0000000000000920

Figure Lengend Snippet: Loss of B4GALT1 mitigates hepatic lipid accumulation according to transcriptome analyses. RNA sequencing was performed on the livers of B4galt1 flox/flox (n=4) and B4galt1 hep−/− (n=3) mice fed the CDAHFD. (A) Gene Ontology analysis of RNA-sequencing results in the liver of B4galt1 flox/flox and B4galt1 hep−/− mice. (B) Volcano plot depiction of significantly upregulated and downregulated genes. (C) Gene Set Enrichment Analysis plot (left) of enrichment in the lipidsynthetic process signature; heatmap (right) of significantly downregulated target genes. Quantitative PCR (D) and immunoblot (E) analysis of lipidsynthetic-related genes or protein expression in liver samples from CDAHFD-fed mice. (F) Gene Set Enrichment Analysis plot of enrichment in the fatty acid β-oxidation signature. (G) Quantitative PCR analysis of hepatic mRNA expression involved in fatty acid oxidation and lipid transport. (H) Representative images and quantitative analysis of Oil Red O staining in si-NC and si B4galt1 cells treated with 0.5% BSA or FFA (0.5 mM) for 24 hours. Scale bar, 100 µm. Data are presented as mean ± SEM. For (D) and (G), significance was determined by the Student t test. For (H), significance was determined by the ANOVA (* p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001). Abbreviations: B4GALT1, β-1,4-galactosyltransferase 1; BSA, bovine serum albumin; CDAHFD, choline-deficient, L–amino acid–defined, high-fat diet; FFA, free fatty acids.

Article Snippet: Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and non-esterified fatty acids (NEFA) as well as the TG, TC, LDL, and NEFA levels in murine liver tissues were quantified following standardized manufacturer protocols (ALT: cat. no. C009-2; AST: cat. no. C010-2; TG: cat. no. A110-1; TC: cat. no. A111-1; LDL: cat. no. A113-1; NEFA: cat. no. A042-2; Nanjing Jiancheng).

Techniques: RNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining

Journal: Hepatology Communications

Article Title: B4GALT1 deficiency attenuates steatohepatitis by regulating the PPARγ/ACSL4 axis

doi: 10.1097/HC9.0000000000000920

Figure Lengend Snippet: B4GALT1 genetic modulation orchestrates lipid–induced hepatocyte ferroptosis in experimental models. (A) Representative TEM images of the murine model. White arrowheads denote damaged mitochondria (shrunken or inflated mitochondria, reduced number of mitochondrial cristae, and destroyed mitochondrial membrane). Scale bars, 500 nm. (B) Gene Set Enrichment Analysis plot (left) of enrichment in the ferroptosis signature; heatmap (right) of key genes. Quantitative PCR (C) and immunoblot (D) analysis of ferroptosis-related genes or protein expression in liver samples from CDAHFD-fed mice. (E) Level of MDA (n=6/group) and ratio of GSH to GSSG (n=5–6/group) in B4galt1 flox/flox and B4galt1 hep−/− mice fed a chow diet or CDAHFD. (F–H) AML12 cells were transfected with si-NC or B4galt1 siRNA, and vector or B4galt1 overexpressed (OE) plasmid for 48 hours, and then treated with 0.5% BSA or FFA (0.5 mM) for 24 hours. (F) Level of MDA and ratio of GSH to GSSG (n=6/group). (G) Representative images of C11-BODIPY staining and quantified mean fluorescence intensity of C11-BODIPY images (n=5/group). Scale bar, 50 µm. (H) Immunoblots analysis of ACSL4, SLC7A11, and GPX4. Data are presented as mean ± SEM. For (C), significance was determined by the Student t test. For (E), (F), and (G), significance was determined by the ANOVA (* p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001). Abbreviations: B4GALT1, β-1,4-galactosyltransferase 1; BSA, bovine serum albumin; CDAHFD, choline-deficient, L–amino acid–defined, high-fat diet; FFA, free fatty acids; GSH, glutathione; GSSG, glutathione disulfide; MDA, malondialdehyde.

Article Snippet: Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and non-esterified fatty acids (NEFA) as well as the TG, TC, LDL, and NEFA levels in murine liver tissues were quantified following standardized manufacturer protocols (ALT: cat. no. C009-2; AST: cat. no. C010-2; TG: cat. no. A110-1; TC: cat. no. A111-1; LDL: cat. no. A113-1; NEFA: cat. no. A042-2; Nanjing Jiancheng).

Techniques: Membrane, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation, Staining, Fluorescence

Journal: Hepatology Communications

Article Title: B4GALT1 deficiency attenuates steatohepatitis by regulating the PPARγ/ACSL4 axis

doi: 10.1097/HC9.0000000000000920

Figure Lengend Snippet: B4GALT1 deficiency suppresses lipid peroxidation via a PPARγ/ACSL4-dependent mechanism. (A–C) AML12 cells were co-transfected with si-NC or B4galt1 siRNA combined with vector or Acsl4 overexpressed (OE) plasmid for 48 hours and then exposed to FFA (0.5 mM) for 24 hours. (A) Level of MDA and ratio of GSH to GSSG (n=6/group). (B) Representative images of C11-BODIPY staining. Scale bar, 50 µm. (C) Quantified mean fluorescence intensity of C11-BODIPY images (n=5/group). (D) Immunoblot analysis and ACSL4 immunoprecipitation analysis evaluating RCA-I in AML12 cells. (E) Immunoblot analysis of ACSL4 in AML12 cells transfected with vector or B4galt1 OE for 48 hours prior to exposure to 0.5% BSA or FFA together with DMSO or pioglitazone (10 μm) for 24 hours. (F) Immunoblot analysis of ACSL4 in AML12 cells co-transfected with vector or B4galt1 OE combined with Acsl4 OE plasmid for 48 hours prior to exposure to 0.5% BSA or FFA for 24 hours. (G) Prediction of PPARγ binding sites to the Acsl4 promoter in the Jaspar database and the corresponding primer sequences. (H) Chromatin immunoprecipitation assays were performed in AML12 cells transfected with si-NC or B4galt1 siRNA prior to exposure to 0.5% BSA or FFA using antibodies against PPARγ and IgG. Data are presented as mean ± SEM. For (A), (C), and (H), significance was determined by the ANOVA (** p <0.01, *** p <0.001, **** p <0.0001). Abbreviations: ACSL4, acyl-CoA synthetase long chain family member 4; B4GALT1, β-1,4-galactosyltransferase 1; BSA, bovine serum albumin; FFA, free fatty acids; PPARγ, peroxisome proliferator-activated receptor gamma; RCA-I, Ricinus communis agglutinin I.

Article Snippet: Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and non-esterified fatty acids (NEFA) as well as the TG, TC, LDL, and NEFA levels in murine liver tissues were quantified following standardized manufacturer protocols (ALT: cat. no. C009-2; AST: cat. no. C010-2; TG: cat. no. A110-1; TC: cat. no. A111-1; LDL: cat. no. A113-1; NEFA: cat. no. A042-2; Nanjing Jiancheng).

Techniques: Transfection, Plasmid Preparation, Staining, Fluorescence, Western Blot, Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation

Journal: Hepatology Communications

Article Title: B4GALT1 deficiency attenuates steatohepatitis by regulating the PPARγ/ACSL4 axis

doi: 10.1097/HC9.0000000000000920

Figure Lengend Snippet: B4GALT1 interacts with PPARγ and regulates the protein stability of PPARγ through N-glycosylation. (A) Immunoblot analysis of PPARγ levels in AML12 cells transfected with B4galt1 siRNA versus si-NC (left) and in AML12 cells transfected with B4galt1 -overexpressing (OE) versus vector (right) exposure to either 0.5% BSA or FFA. (B) Immunoblot analysis of PPARγ levels in liver samples taken from B4galt1 flox/flox and B4galt1 hep−/− mice fed CDAHFD (n=3/group). (C) Cycloheximide chase assay of PPARγ expression in AML12 cells transfected with B4galt1 siRNA versus si-NC (top) and in B4galt1 -overexpressing AML12 cells versus empty vector - transfected cells (bottom). (D) Co-immunoprecipitation analysis evaluating the interaction between B4GALT1 and PPARγ in AML12 cells using exogenous Flag-B4galt1 or Myc-Pparγ, Flag-vector, or Myc-vector as a control. (E) Immunoblot analysis of PPARγ levels in B4galt1 -knockdown AML12 cells, B4galt1 -overexpressing AML12 cells, and their corresponding si-NC/vector cells after treatment with tunicamycin (TM) and FFA. (F) Immunoblot and PPARγ immunoprecipitation analyses of RCA-I levels in AML12 cells transfected with si-NC or B4galt 1 siRNA in combination with Myc-vector or Myc-Pparγ plasmid. (G–I) AML12 cells were co-transfected with vector or B4galt1 OE in combination with Acsl4 OE plasmid for 48 hours and then exposed to FFA for 24 hours. (G) Levels of MDA and the ratio of GSH to GSSG (n=6/group). (H) Representative images of C11-BODIPY staining. Scale bar, 50 µm. (I) Quantified mean fluorescence intensity of C11-BODIPY images (n=5/group). Data are presented as mean ± SEM (* p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001; ANOVA). Abbreviations: B4GALT1, β-1,4-galactosyltransferase 1; BSA, bovine serum albumin; FFA, free fatty acids; PPARγ, peroxisome proliferator-activated receptor gamma; RCA-I, Ricinus communis agglutinin I; TM, tunicamycin.

Article Snippet: Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and non-esterified fatty acids (NEFA) as well as the TG, TC, LDL, and NEFA levels in murine liver tissues were quantified following standardized manufacturer protocols (ALT: cat. no. C009-2; AST: cat. no. C010-2; TG: cat. no. A110-1; TC: cat. no. A111-1; LDL: cat. no. A113-1; NEFA: cat. no. A042-2; Nanjing Jiancheng).

Techniques: Glycoproteomics, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Control, Knockdown, Staining, Fluorescence

Journal: Comprehensive Physiology

Article Title: Systemic Consequences of Chronic Ethanol Intake: From Microbiome Shifts to Metabolic Impairment

doi: 10.1002/cph4.70132

Figure Lengend Snippet: Chronic EtOH induces gut permeability, hepatic injury & inflammation. (A, B) Indirect makers of intestinal barrier dysfunction: Serum LPS and zonulin. (C, D) Serum markers of hepatic injury: ALT and AST. (E–I) Liver pro‐inflammatory markers: TNFα and IL‐6 protein expression, and the acute‐phase protein serum amyloid A quantified using a colorimetric ELISA (Panel H; gray outlines indicate standards; blue, control; orange, EtOH). Statistical analyses included unpaired t ‐tests or Welch's t ‐tests. *Significantly different from control group ( p < 0.05). Data are presented as mean ± SEM. Samples sizes were 6–7 for controls and 8–9 for EtOH.

Article Snippet: Free fatty acids (FFAs) in subcutaneous WAT were quantified using an enzymatic colorimetric assay kit (Cat # E‐BC‐K013‐S, Elabscience Laboratories).

Techniques: Permeability, Expressing, Enzyme-linked Immunosorbent Assay, Control