Journal: Science Advances

Article Title: Atf3-mediated metabolic reprogramming in hepatic macrophage orchestrates metabolic dysfunction–associated steatohepatitis

doi: 10.1126/sciadv.ado3141

Figure Lengend Snippet: ( A and B ) Lv-Atf3 , Atf3 M−/− , or control mice were fed a WD. After 20 weeks, HSCs from the mice were isolated and relative mRNA levels of genes involved in fibrogenesis were determined by qRT-PCR ( n = 3 per group). ( C and D ) Hepatic macrophages from Atf3 fl/fl or Atf3 M−/− mice (fed a chow diet) were isolated, and total RNA was extracted for RNA-seq ( n = 3). Some biological processes were regulated by Atf3 (C). The heatmap shows that some cytokines were up-regulated or down-regulated by Atf3 (D). ( E ) mRNA levels. ( F ) Protein levels. ( G and H ) Atf3 fl/fl or Atf3 M−/− mice were fed a WD. After 12 weeks, liver macrophages were isolated and cultured for 24 hours. Then, the media were collected and cocultured with freshly isolated hepatocytes (G) or HSCs (H) in the presence of anti-Rbp4 (10 mg/ml). After 12 hours, relative mRNA levels of genes involved in fibrogenesis were determined. ( I and J ) Liver macrophages from C57BL/6J mice were isolated and then infected with Lv-Gfp or Lv-Atf3 for 24 hours. The mRNA or protein levels of Rbp4 were measured. ( K and L ) Lv-Gfp or Lv-Atf3 liver macrophages were cultured for 24 hours. The media were collected and cocultured with primary hepatocytes (K) or mouse HSCs (L) in the presence of recombinant mouse Rbp4 protein or vehicle (5 μg/ml). The mRNA levels of hepatocytes or HSCs were determined. ( M and N ) Primary hepatocytes or mouse HSCs were infected with lentiviruses expressing shRNA against scramble sequences (Lv-shScr) or Stra6 (Lv-shStra6) for 24 hours and then incubated with recombinant mouse Rbp4 protein or vehicle (5 μg/ml). The mRNA levels of hepatocytes (M) or HSCs (N) were measured. * P < 0.05; ** P < 0.01. NS, not significant.

Article Snippet: Recombinant mouse Rbp4 protein (catalog no. 3476-LC-050) and recombinant mouse FGF-21 protein (catalog no. 8409-FG-025/CF) were purchased from R&D Systems.

Techniques: Control, Isolation, Quantitative RT-PCR, RNA Sequencing Assay, Cell Culture, Infection, Recombinant, Expressing, shRNA, Incubation

Journal: Science Advances

Article Title: Atf3-mediated metabolic reprogramming in hepatic macrophage orchestrates metabolic dysfunction–associated steatohepatitis

doi: 10.1126/sciadv.ado3141

Figure Lengend Snippet: ( A to H ) Hepatic macrophages from Rbp4 M−/− mice were isolated and then infected with Lv-Gfp or Lv-Atf3 for 24 hours. The macrophages were transplanted into C57BL/6J male mice and then fed a WD for 18 weeks ( n = 6 per group). (A) Hepatic macrophage Atf3 protein levels. (B) Body weight and liver weight. (C) Plasma ALT/AST. (D) Hepatic lipid levels. (E) Hepatic FFA levels. (F) Hepatic hydroxyproline levels. (G) Representative liver section images stained by Oil Red O or picrosirius red. (H) Hepatic mRNA levels. ( I ) C57BL/6J mice were fed chow diet or WD fed for 8 or 16 weeks. Hepatic macrophages were isolated, and Rbp4 protein levels were determined. ( J ) Plasma Rbp4 protein levels of Lv-Atf3 mice or Atf3 M−/− mice were measured. ( K to S ) Atf3 M−/− mice were fed a WD for 13 weeks, and the mice were intraperitoneally (i.p.) injected with 100 μg of anti-Rbp4 antibodies (per mouse) or vehicle once every 6 days for 30 days ( n = 6 per group). The experimental procedures are illustrated in (K). The levels of hepatic lipids (L), FFAs (M), hydroxyproline (N), and plasma ALT/AST were analyzed. (O) Representative liver section images stained by H&E or picrosirius red. (P) MASH score. (Q) Fibrotic area. (R) Plasma ALT/AST. (S) Hepatic mRNA levels. ( T ) Simplified model depicting the role of hepatic macrophage Atf3 in regulation of MASH in mice. * P < 0.05; ** P < 0.01.

Article Snippet: Recombinant mouse Rbp4 protein (catalog no. 3476-LC-050) and recombinant mouse FGF-21 protein (catalog no. 8409-FG-025/CF) were purchased from R&D Systems.

Techniques: Isolation, Infection, Staining, Injection