Oral administration of I3A alleviates diet induce hepatic steatosis and inflammation.

(A) Fecal, (B) liver and (C) serum concentrations of I3A in male B6 129SF1/J mice in control low-fat diet (CN), Western diet (WD), Western diet with low-dose I3A (WD-50), and Western diet with high-dose I3A (WD-100) at week 16. (D) Serum alanine aminotransferase (ALT) levels in mice at weeks 10 and 16. (E) Liver triglyceride (TG) levels at week 16. Data shown are TG concentrations (mg/dl) normalized to corresponding tissue DNA contents (µg DNA). (F) Representative liver sections stained with hematoxylin-eosin (H&E). (G) Histology score for steatosis, hepatocyte ballooning and lobular inflammation. H&E-stained liver sections were evaluated by an expert pathologist using the NASH CRN and fatty liver inhibition of progression (FLIP) consortia criteria. Data shown are mean ± SEM (n = 10 per group). *: p<0.05, **: p<0.01, ***: p<0.001 using Wilcoxon rank sum test.

I3A administration reverses WD induced alterations in liver inflammatory cytokines and bile acids.

(A) Inflammatory cytokines in liver tissue at week 16. (B) Liver total bile acid concentration (left panel), and abundance of CDCA branch bile acids relative to total bile acids pool (right panel). Data shown are mean ± SEM. *: p<0.05, **: p<0.01, ***: p<0.001 using Wilcoxon rank sum test.

I3A administration partially reverses diet-induced metabolome alterations in the liver.

(A) Scatter plots of latent variable projections from PLS-DA of untargeted metabolomics data features. Comparison of all four experimental groups (left panel), CN vs. WD group (middle panel), and WD vs. WD-50 and WD-100 groups (right panel). (B) Heatmap of significant metabolite features (FDR<0.1) based on statistical comparisons of treatment groups (CN vs. WD). (C) KEGG pathway enrichment analysis of the metabolites. Number in the parenthesis represents the metabolites detected in the pathway. (D) Schematic for tryptophan metabolism (left panel). Tryptophan metabolism metabolites fold changes of WD-50, WD-100, CN relative to WD (right panel). (E) Acyl-Carnitine fold change of WD-50, WD-100, CN relative to WD. p-values were calculated using Student t-test and corrected by FDR.

I3A administration partially reverses diet-induced proteome alterations in the liver.

(A) Scatter plots of latent variable projections from PLS-DA of confidently identified proteins. Comparison of all four experimental groups (left panel), CN vs WD group (middle panel), and WD vs. WD-50 and WD-100 groups (right panel). (B) Heatmap of significant proteins having Variable Importance in Projection score >1.2. The proteins were clustered using k-means. (C) Pathway enrichment analysis of significant proteins differentially abundant in CN vs. WD comparison (upper panel) and WD-100 vs. WD comparison (lower panel). GeneRatio divides the number of significantly altered proteins that are in the pathway by the total number of significantly altered proteins. BGRatio divides the number of proteins that are in the pathway by the number of all detected proteins. The p-value was calculated using Fisher’s exact test.

I3A administration reduces the levels of enzymes in fatty acid transport, de novo lipogenesis and β-oxidation.

(A) Abundance of fatty acid translocase (CD36) and fatty acid synthase. (B) Mitochondrial and (C) peroxisomal fatty acid oxidation enzymes. Data shown are mean ± SEM. *: p<0.05, **: p<0.01 using Wilcoxon rank sum test

I3A modulates AMPK phosphorylation and suppresses RAW264.7 macrophage cell inflammation in an AMPK dependent manner.

(A) and (B) I3A administration reverses WD induced reduction in liver p-AMPK and AMPK. (A) Levels of p-AMPK and AMPK in liver tissue at week 16 as determined by Western blot analysis. (B) Ratios of p-AMPK (left panel) and AMPK (right panel) to β-actin. The ratios were determined based on the p-AMPK and AMPK band intensities quantified using Image Lab (Bio-Rad) and normalized to the loading control (β-actin). Data shown are mean ± SEM. *: p<0.05, **: p<0.01 using Wilcoxon rank sum test. (C) Expression levels of p-AMPK and total AMPK in RAW 264.7 macrophages pre-treated with either I3A or vehicle (DMF) control followed by stimulation with palmitate and LPS, determined by Western blot analysis. (D) Fold-changes in p-AMPK and total AMPK. Fold-changes were calculated relative to the DMF and no palmitate and LPS stimulation condition. The band intensities were quantified and normalized to loading control (β-actin) by using Image Lab (Bio-Rad). (E) Expression levels of tnfα and il-1β in RAW 264.7 cells treated with p-AMPK activator AICAR, followed by stimulation with palmitate and LPS. (F) Expression levels of tnfα (top row) and il-1β (bottom row) in RAW 264.7 cells transduced with non-targeted control siRNA (left panels) or Prkaa1 siRNA (middle panels), pre-treated with I3A, and then stimulated with palmitate and LPS. Data shown are mean ± SEM from three independent cultures with three biological replicates. *: p<0.05, **: p<0.01, ***: p<0.001 using Student’s t-test.

LC-MS parameters for bile acid analysis

LC-MS parameters for free fatty acid analysis

Gradient method for untargeted metabolomics

Gradient method for bile acid analysis

Gradient method for free fatty acid analysis

Gradient method for untargeted proteomics

LC-MS parameters for targeted proteomics

Primer sequences

Study design.

(A) Three groups of male B6 129SF1/J mice (n=10 for each group) were fed ad libitum a Western diet (WD) and a sugar water (SW) solution while a fourth group was given normal chow diet. After 8 weeks, the three groups of WD-fed mice were randomly selected for treatment with vehicle (WD group) or low (WD-50 group) or high dose (WD-100 group) of I3A for an additional 8 weeks. The fourth group (CN) was continued on low-fat diet calorie matched with the WD. (B) Sampling scheme. (C) Quantification of I3A in feces at week 8. (D) Body weight increase and food intake of the four groups. Body weights were normalized to week 8 body weight when I3A administration was started. Food intakes were measured per cage and average food intake was calculated as per gram food intake per mouse per day. Data shown are mean ± SEM from a replicate study. *: p<0.05, **: p<0.01, WD group compared to CN group using Wilcoxon rank sum test.

I3A reduces liver inflammatory cytokine expression and total FFA concentration.

(A) Inflammatory cytokines in liver tissue at week 16. (B) FFAs in diet, serum and liver samples. Data shown are mean ± SEM. *: p<0.05, **: p<0.01, ***: p<0.001 using Wilcoxon rank sum test.

I3A administration does not significantly alter the fecal microbial community.

(A) Alpha diversity of the fecal microbiome from CN, WD, WD-50, and WD-100 groups. (B) Analysis of Similarities (ANOSIM) comparison for CN vs. WD group and WD vs. WD-50 vs. WD-100 groups. (C) Phylum and (D) genus level relative abundance of the fecal microbial community members. (E) LEfSe results at the genus level. *: p<0.05, ***: p<0.001 using Wilcoxon rank sum test.

I3A administration does not significantly alter the fecal metabolome.

(A) Score plots show the first two principal components for all four experimental groups (left panel), CN vs. WD group (middle panel), and WD vs. WD-50 and WD-100 groups (right panel). Numbers in the parentheses of axis titles show percent of variance explained by the corresponding principal component. Ellipses circumscribe 95% confidence regions for the experimental groups assuming Gaussian distribution of component scores. (B) Heatmap of fecal microbiome metabolite features detected in all treatment groups. Rows and columns are features and treatment groups, respectively. The features were clustered using k-means.

Principal Component Analysis of liver proteome after I3A administration.

(A) Score plots of the first two principal components for all four experimental groups (left panel), CN vs. WD group (middle panel), and WD vs. WD-50 and WD-100 groups (right panel). Numbers in the parentheses of axis titles show percent of variance explained by the corresponding principal component. Ellipses represent 95% confidence regions for the experimental groups assuming Gaussian distribution of component scores.

I3A administration reduces the levels of antioxidant enzymes and affects iron metabolism related proteins.

(A) Catalase and glutathione peroxidase-1 abundance for the four experimental groups. (B) Hepcidin and Serotransferrin abundance. Data shown are mean ± SEM. *: p<0.05, **: p<0.01 using Wilcoxon rank sum test.

I3A’s anti-inflammatory effects in macrophages are independent of AhR activation.

A. Expression level of AhR in RAW264.7 and AML12 cells determined by Western blot analysis. B. Raw 264.7 cells were treated with 1mM I3A (or DMF solvent control) for 4h, then stimulated with 300µM Palmitate for 18h and 10ng/ml LPS for 6h (two-hits model). The AhR inhibitor CH223191 (5µM) or DMSO control were added 10min before I3A treatment. Total RNA were isolated from the cells and the expression of tnfα and il-1β were measured with qRT-PCR. C. tnfα and il-1β expression were plotted as fold change normalized to the DMF control group. Data presented as the mean ± SEM. ***: p<0.001 using Student’s t-test.

Prkaa1 siRNA reduces AMPK expression in macrophages.

(A) Levels of prkaa1 mRNA in RAW 264.7 cells transfected with prkaa1 or non-targeted control siRNA for 24 h, followed by incubation for an additional 24, 48, and 72 h. The expression level of prkaa1 is normalized to that of the housekeeping gene β-actin. (B) Western blot analysis of p-AMPK and total AMPK from cells treated with the different siRNA. A representative blot is shown. (C) Quantified intensities of p-AMPK and total AMPK bands normalized to loading control (β-actin).

Proposed model for the effects of I3A on hepatic lipid metabolism and inflammation.

When mice are fed with a WD (top panel), TG and FFAs accumulate in the liver due to increased uptake of fatty acids. This also leads to increased β-oxidation in the mitochondria and peroxisomes. In liver macrophages, the increase in FFAs, possibly in conjunction with circulating endotoxins (e.g., LPS) stimulate production of inflammatory cytokines. When mice fed the WD are treated with I3A (bottom panel), both TG and FFAs decrease in the liver. Rather than impact fatty acid uptake, I3A treatment reduces de novo lipogenesis through a downregulation of Fasn, while also reducing both mitochondrial and peroxisomal β-oxidation. In macrophages, I3A attenuates fatty acid and LPS stimulated production of inflammatory cytokines through activation of AMPK.

Untargeted proteomic data analysis workflow.

The individual steps (A - G) are described in the untargeted proteomics section of Supplemental Information.