Figures and data
AOAH reduces hepatic lipid accumulation.
(A) AOAH cleaves the two piggyback fatty acyl chains from the lipid A moiety, inactivating LPS. The arrows indicate the cleavage sites.
(B) Co-housed Aoah+/+ and Aoah−/− mice were fed either a normal diet (NC) or a high fat diet plus high fructose (23.1 g/L) and glucose (18.9 g/L) in their drinking water (HFD) for 28 weeks.
(C) Body weight was measured weekly for 28 weeks. Data were combined from 4 experiments. n = 12 – 17.
(D) Representative images of livers at 28 weeks are shown and the liver weight was measured. n = 7 – 9.
(E) Mouse livers were fixed, sectioned and stained with Oil Red O or Hematoxylin-eosin (H & E). In Oil Red O-stained sections, the percentage of area occupied by the lipid droplets was quantified using Image J. H & E staining results were semi-quantitatively scored for disease severity. Data were combined from 3 experiments, n = 6.
(F) Triacylglycerol (TAG) and total cholesterol (TCHO) were measured in liver homogenates. Data were combined from at 3 experiments, n = 9 – 11.
(G) The serum concentrations of triglyceride, total cholesterol, HDL, LDL and free fatty acids were measured. Data were combined from 3 experiments. n = 6 – 10.
(C - G) Mann-Whitney test and Two-way ANOVA test were used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
AOAH prevents hepatic inflammation and tissue injury when mice are fed HFD.
(A) Serum ALT and AST were measured. Data were combined from 3 experiments, n = 7 – 12.
(B-C) Inflammation-related Tnf-α, Il-6, Ifn-γ, Il-10 mRNA and fibrosis-related Timp1, Mmp2, α-SMA, Collagen 4α1 mRNA were measured in livers at 28 weeks. Data were combined from 3 experiments, n = 6 – 10.
(D) Gating strategy to identify hepatic neutrophils, monocytes, Kupffer cells, lipid-associated macrophages (LAM) and monocyte-derived Kupffer cells (Mo-KC) subsets.(E) The myeloid cell numbers in Aoah+/+ and Aoah−/− livers were calculated using FACS analysis. Data were combined from 3 experiments, n = 6 – 8. Mann-Whitney test was used. *, P < 0.05; **, P < 0.01.
AOAH reduces hepatic LPS levels and the expression of fatty acid uptake and synthesis genes.
(A) AOAH expression in different kinds of liver cells based on single cell analysis by Remmerie et al.(Remmerie et al., 2020) is shown.
(B) Kupffer cells, monocytes and NK cells were sorted using flow cytometry and hepatocytes were purified from 6 - 8 weeks old mice. AOAH mRNA was measured. Data were combined from 3 experiments, n = 7 – 8.
(C) We confirmed that AOAH protein was present in 6 - 8-week-old Aoah+/+ mouse livers but not in Aoah−/−mouse liver homogenates using Western blot. Representative pictures from 3 experiments were shown.
(D) Heated feces suspension, liver homogenates and plasma from Aoah+/+ and Aoah−/−mice were tested for TLR4-stimulating activity. Data were combined from at least 3 experiments, n = 6 – 10.
(E) Gut permeability was measured. Mice were fasted for 18 h. Mice were orally gavaged with fluorescein isothiocyanate (FITC)-conjugated dextran and 4 h later, FITC fluorescence was measured in plasma. Data were combined from 3 experiments, n = 5 – 7.
(F) At 28 weeks of NC or HFD feeding, liver Fabp3 and Fasn mRNAs were measured. Data were combined from 3 experiments, n = 8 – 11.
Mann-Whitney test was used. *, P < 0.05; **, P < 0.01.
AOAH regulates the expression of hepatic fatty acid metabolism genes.
(A-B) Co-housed 6 – 8 weeks old (i.e. young) Aoah+/+ and Aoah−/−mouse livers were subjected to RNAseq analysis. The differentially expressed genes were shown in the volcano plot (A). Fatty acid biosynthesis pathway was found enriched using GSEA (Gene set enrichment analysis) (B, left panel). The fatty acid biosynthesis-associated gene expression levels of co-housed Aoah+/+ and Aoah−/− mouse livers are shown as a heatmap, n = 4 (B, middle panel). The fatty acid biosynthesis pathway is shown. The enzymes marked red had increased expression in young Aoah−/− mouse livers (B, right panel).
(C-E) The hepatic expression of fatty acid synthesis (C), uptake (D) and oxidation (E) genes was measured in co-housed 6 - 8 weeks old Aoah+/+and Aoah−/− mice. Data were combined from 3 experiments, n = 5 – 8.
Mann-Whitney test was used. *, P < 0.05; **, P < 0.01.
AOAH reduces hepatic SREBP1.
(A) SREBP-1a and SREBP-1c gene mRNA was measured in livers from Aoah+/+ and Aoah−/− mice. n = 8.
(B) Liver homogenates from Aoah+/+ and Aoah−/− mice were subjected to Western analysis. SREBP1 protein levels were quantitated using Image J. flSREBP1 is full length SREBP1, which is a precursor; nSREBP1is nuclear SREBP1, which is an active form. n = 4 – 6.
(C) Liver cytosol and nuclei were separated from Aoah+/+ and Aoah−/− mice and then subjected to Western analysis. SREBP1 protein levels were quantitated using Image J. n = 6.
Mann-Whitney test was used. *, P < 0.05; **, P < 0.01.
Excessive gut-derived LPS increases hepatic nSREBP1 and mTOR activation.
Livers from Aoah+/+mice, Aoah−/− mice and Aoah+/+ mice that were orally gavaged (i.g.) with 200 μg LPS 24 h earlier were obtained.
(A) LPS levels were measured.
(B) The mRNA levels of fatty acid biosynthesis genes and CD36 were measured using qPCR.
(C) SREBP1 protein levels and AKT-mTOR activities were measured using Western and Image J. Livers from Aoah−/− mice and Aoah+/+ mice that were orally gavaged (i.g.) with 200 μg LPS 24 h earlier had increased nSREBP1 and mTOR activities than did those from control Aoah+/+ mice.
Data were combined from 3 experiments, n = 3 – 7.
Mann-Whitney test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
AOAH prevents NAFLD by inactivation of gut-derived LPS.
Gut-derived LPS may translocate via the portal vein to the liver. In Aoah+/+ mice, LPS is deacylated in the intestine and portal venous blood; when LPS reaches the liver, LPS is further inactivated by hepatic AOAH. In Aoah−/− mouse liver, gut-derived LPS cannot be deacylated and remains stimulatory. LPS stimulates hepatocytes to generate nuclear SREBP1, which promotes fatty acid biosynthesis gene expression. LPS also increases the expression of fatty acid uptake genes CD36 and Fabps while reducing that of fatty acid oxidation related genes, Acot2 and Ppar-α. As a result of persistent LPS stimulation, Aoah−/− mice are more susceptible to NAFLD than are Aoah+/+ mice. dLPS = deacylated LPS.
