Figure 8. Wnt signaling is reduced in fibrotic livers of NcDase^−/− mice.

(A-D) Livers of HFD-induced NASH NcDase^−/− mice and WT mice were examined.

(A) Real time PCR analysis for the expression of Wnt ligands and receptors in livers.

(B) Immunoblotting analysis of Wnt signal in the liver.

(C) Real-time PCR analysis for the expression of Wnt target genes.

(D-E) Primary hepatocytes were treated with control media, or media from L-Wnt3a with/without a final concentration of 200 μM BSA-conjugated fatty acids or vehicle control for 12h-48h.

(D) Immunofluorescence staining of β-catenin in hepatocytes.

(E) Immunoblotting analysis of Wnt signal in hepatocytes 24h after treatment.

(F) Real time PCR analysis for the mRNA levels of NcDase, SCD1, SCD2 and CCND1 12h-24h after treatment in primary hepatocytes from WT mice.

(G) The activity of NcDase in primary WT 24h after Wnt3a treatment.

(H) Primary hepatocytes were treated with DMSO or Wnt3a inhibitor ICG-001 for 24h. Real-time PCR analysis for the mRNA levels of indicated target genes.

(I) Primary hepatocytes from NcDase^−/− or WT mice were treated with control or PA+Wnt3a for 24h. Real-time PCR analysis for the mRNA levels of indicated target genes.

Mean ± SEM; n = 7. *p < 0.05, **p < 0.01.

(J) Masson’s trichrome (top) and H&E (bottom) staining of liver sections. Four groups of mice, including WT mice injected with control adenovirus (WT-Adv), WT mice injected with SCD1 adenovirus (WT-Adv-SCD1), NcDase^−/− mice injected with control adenovirus (NcDase^−/−-Adv), NcDase^−/− mice injected with SCD1 adenovirus (NcDase^−/−-Adv-SCD1), were fed with HSPC for 16 weeks. n=5.

(K) Schematic diagrams of how NcDase regulates the SCD1-Wnt/β-catenin loop to protect from NASH via gut IgA associated microbiota. NcDase regulates IgA production, which appropriately affects the expansion of Desulfovibrio and the colonization of beneficial Ruminococcaceae family. Ruminococcaceae function to temper expression of SCD1 and MUFAs production. MUFAs promotes Wnt fatty acylation and β-catenin activation, which leads to NASH. Abbreviations: AXIN2, axis inhibition protein 2; EV, extracellular vesicle; FZD6, frizzled homolog 6 (Drosophila); GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jag1, jagged canonical Notch ligand 1; Myc, MYC proto-oncogene; OA, Oleic acid; p-AMPK, AMP-activated protein kinase phosphorylation.