A retinoic acid receptor β2 agonist attenuates transcriptome and metabolome changes underlying nonalcohol-associated fatty liver disease

Figures

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  Figure 1. Disease signature pathway analysis shows that AC261066 limits/prevents high-fat diet (HFD)-induced changes in the transcripts underlying development of NAFLD.A, volcano plot showing total numbers of transcripts with statistically significant increases or decreases (q < 0.1) in the HFD compared with the chow group (n = 6 per group). B, volcano plot showing total numbers of genes with statistically significant increases or decreases (q < 0.1) in the HFD+AC261066 compared with the HFD group (n = 6 per group). C, disease signature pathways increased in the HFD/chow. D, disease signature pathways decreased in the HFD+AC261066/HFD.

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  Figure 2. Transcript and protein levels of NAFLD driver genes are increased in the HFD compared with the chow group, and AC261066 (AC261) ameliorates these changes.A, heatmap analyses of transcript levels of NAFLD driver genes from individual samples in all groups (n = 6 per group). B, the same data as in (A), but with statistical significance shown. Quantitative comparison of mRNA levels of the NAFLD diver genes from the RNA-seq data. The y axes (relative mRNA levels) are differentially expressed gene (DEG) transcript levels (n = 6 per group). C, immunoblotting of the hepatic protein levels of PKLR, FASN, and THRSP (Cropped images). Each lane is a sample from one mouse. Multiple Westerns were run to obtain quantitative data in (D). D, quantification of immunoblotting data (C). E, immunohistochemical analysis of FASN protein (arrows) in the liver sections from all groups. Scale bar = 50 μm. HFD+AC261=HFD+AC261066. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, compared with the HFD group.

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  Figure 3. HFD induced increases in the genes involved in hepatic lipogenesis and AC261066 (AC261) limits these increases.A, heatmap analyses of the transcript levels of genes involved in lipogenesis among all groups (n = 6 per group). B, the same data as in (A), but with statistical significance shown. Quantitative comparison of mRNA levels of lipogenesis related genes from the RNA-seq data. The y axes (relative mRNA levels) are differentially expressed gene (DEG) transcript levels (n = 6 per group). C, immunoblotting analysis of the hepatic protein levels of CD36 and PPARγ (Cropped images). Each lane is a sample from one mouse. D, quantification of the immunoblotting data from six mice/group (C). E, immunohistochemical analysis of CD36 protein (arrow) in the liver sections from all experimental groups. Magnification is 200×; scale bar is 50 μm. HFD+AC261=HFD+AC261066. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, compared with the HFD group.

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  Figure 4. HFD promotes fructose metabolism in the liver and AC261066 attenuates this effect.A, quantitative comparison of transcript levels of the genes involved in fructose uptake and metabolism from the RNA-seq data. The y axes (relative mRNA levels) are differentially expressed gene (DEG) transcript levels (n = 6 per group). B, immunoblotting analysis of the hepatic protein levels of KHK (Cropped images). Each lane is a sample from one mouse. C, quantification of the KHK immunoblotting data. D, the hepatic levels of metabolites involved in fructose metabolism in all experimental groups (n = 4 per group with two repeats). E, the hepatic levels of metabolites involved in oxidative stress in all experimental groups (n = 4 per group with two repeats). F, AML12 cells were stained with Bodipy reagent to label intracellular lipids after various treatments (Experimental procedures) for 48 h. Quantification of the staining with Fiji (ImageJ) representing the integrated density normalized/number of nuclei (NucBlue)/field. Representative areas highlighted in the insets. Scale bar for the images = 100 μm. Scale bar for the insets = 50 μm. HFD+AC261=HFD+AC261066. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p <…

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  Figure 5. AC261066 does not attenuate lipidaccumulation in RARβ knockout HepG2 cells.A, scheme of the Crispr/Cas9 technology strategy indicating the four guide RNAs (gRNAs) designed in exon 2 of the human RARβ gene. B, Sanger sequencing showing successful editing of one monoclonal colony identified in the gRNA highlighted in blue. C, next-generation sequencing (Deep Sequencing) performed to verify the mutations present in the Crispr/Cas9-edited cells. D, RARB and RARB2 mRNA levels measured in parental (P) and RARβ knockout (KO) HepG2 cells after treatment with 2 μM AC261066 for 72 h compared with vehicle-treated (veh) cells. Experiments were performed two times, each time in duplicate, with six wells/experimental group. E, oleate and palmitate (OL/PL; 2:1; 500 μM) treatment for 48 h in parental (PAR) and RARβ KO HepG2 cells in the presence or absence of AC261066 at a 2 μM concentration. The control groups are treated with the vehicle (0.1% DMSO). Bodipy staining lipid droplets in green and NucBlue staining nuclei in blue. Quantification of the staining with Fiji (ImageJ) representing the integrated density normalized/number of nuclei (NucBlue)/field of three independent experiments. ∗∗∗∗p…

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  Figure 6. HFD alters transcripts of genes involved in the hepatic stellate cell (HSC) secretome and AC261066 ameliorates these changes.A, quantitative comparison of representative transcripts contributing to liver fibrosis from RNA-seq data (n = 6 per group). B, immunohistochemical analysis of Galectin-1 and E-cadherin protein in liver sections from all experimental groups. Scale bar = 50 μm. C and E, quantification of images (B) on the signal intensity and the positively stained area using ImageJ. D, quantitative comparison of representative transcripts contributing to liver inflammation from the RNA-seq data (n = 6 per group). In (A) and (D), the y axes (relative mRNA levels) are differentially expressed gene (DEG) transcript levels. HFD+AC261=HFD+AC261066. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, compared with the HFD group.

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  Figure 7. Model of the effects of HFD and AC261066 in the liver.