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HEP_25798_sm_SuppFig1.tif3600KSupporting Fig. 1. Effects of 4-week ATRA treatment on body weight changes and insulin sensitivity of KK-Ay and ob/ob mice. (A) Body weight changes of KK-Ay (left panel) or ob/ob (right panel) mice from 0 to 4 weeks feeding of normal (open circles) or ATRA-supplemented (filled circles) diets. (B,C) Overall average daily consumption (B) and serum leptin levels (C) of KK-Ay or ob/ob mice for 4 weeks feeding of normal (open columns) or ATRA-supplemented (filled columns) diets. (D,E) Hepatic LEPRa (D), and insulin-like growth factor-binding protein 2 (IGFBP2) (E) mRNA levels relative to those of β-actin in KK-Ay or ob/ob mice for 4 weeks feeding of normal (open columns) or ATRA-supplemented (filled columns) diets. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control diet.
HEP_25798_sm_SuppFig2.tif1983KSupporting Fig. 2. Levels of total IRS1 and phosphorylated IRS1 in HepG2 cells treated with ATRA (24 h) and then leptin (3 h), followed by insulin treatment for 2 min. (A and B) Proteins were detected by Western blot analysis. The densitometric analysis was performed using the public domain NIH ImageJ program. IRS1 (A), and phospho-IRS1 (Y1222) (B). *P < 0.05 between the indicated samples.
HEP_25798_sm_SuppFig3.tif13889KSupporting Fig. 3. Effects of 4-week ATRA treatment on lipid metabolism in mice fed the HFHFr diet. (A,B) Liver-to-body weight ratios (A) and visceral fat weights (B) of control mice (white columns), or NAFLD mice fed for 4 weeks with HFHFr (grey columns) or ATRA + HFHFr (black columns) diets. (C-E) Non-esterified free fatty acids (NEFA) (C), triglyceride (D), and total cholesterol (E) levels in the livers of NAFLD mice of control mice (white columns), or NAFLD mice fed for 4 weeks with HFHFr (grey columns) or ATRA + HFHFr (black columns) diets. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control diet. #P < 0.05; ##P < 0.01; ###P < 0.001 between ATRA + HFHFr die vs. HFHFr diet. (F) Representative hematoxylin and eosin (upper panels) and oil red O (lower panels) staining of liver sections of control mice or NAFLD mice fed HFHFr (grey) or ATRA-supplemented HFHFr (ATRA + HFHFr) diets for 4 weeks (original magnification, 100x).
HEP_25798_sm_SuppFig4.tif1316KSupporting Fig. 4 Effects of 4-week ATRA treatment on insulin sensitivity in mice fed the HFHFr diet. (A,B) Homeostatic model assessment of insulin resistance (HOMA-IR) indices (A) and serum leptin levels (B) of control mice (white columns), or NAFLD mice after 4 weeks feeding HFHFr (grey columns) or ATRA + HFHFr (black columns) diets. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control diet. #P < 0.05; ##P < 0.01; ###P < 0.001 between ATRA + HFHFr die vs. HFHFr diet.
HEP_25798_sm_SuppFig5.tif3673KSupporting Fig. 5. Effects of four-week ATRA treatment on protein expression levels in the livers of mice fed the HFHFr diet. (A-H) Proteins involved in insulin and leptin signaling pathways were detected by Western blot analysis of liver samples from control mice (white columns), or NAFLD mice after 4 weeks feeding HFHFr (grey columns) or ATRA + HFHFr (black columns) diets. IRS1 (A), phospho-IRS1 (Y1222) (B), short LEPR isoform (C), JAK2 (D), phospho-JAK2 (Y1007/1008) (E), STAT3 (F), phospho-STAT3 (Y705) (G), and SOCS3 (H). The densitometric analysis was performed using the public domain NIH ImageJ program. Data are expressed relative to actin levels. **P < 0.01; ***P < 0.001 vs. control diet (white columns). #P < 0.05; ##P < 0.01; ###P < 0.001 between ATRA + HFHFr diet (black columns) vs. HFHFr diet (grey columns).
HEP_25798_sm_SuppFig6.tif2550KSupporting Fig. 6. Effects of 4-week ATRA treatment on the expression levels of RARa and RARβ and the leptin signaling pathway in livers of mice fed the HFHFr diet. (A) Representative images of Western blots of liver samples from control mice (CTRL), or NAFLD mice after 4 weeks feeding of HFHFr or ATRA + HFHFr diets. (B,C) The expression levels of RARa (B) and RARβ (C) proteins in the liver of control mice (white columns), or NAFLD mice after 4 weeks feeding HFHFr (grey columns) or ATRA-supplemented HFHFr (black columns) diets. (D,E) Relative hepatic LEPRa (D), and insulin-like growth factor-binding protein 2 (IGFBP2) (E) mRNA levels to β-actin in control mice (white columns), or NAFLD mice after 4 weeks feeding HFHFr (grey columns) or ATRA-supplemented HFHFr (black columns) diets. *P < 0.05 vs. control diet. #P < 0.05 between ATRA + HFHFr diet vs. HFHFr diet.
HEP_25798_sm_SuppFig7.tif12500KSupporting Fig. 7. Effects of four-week ATRA treatment on STAT3 activity in hepatocytes of mice fed the HFHFr diet. Representative images of STAT3 immunohistochemical staining of liver sections from control mice (upper left panel), or NAFLD mice after 4 weeks feeding of HFHFr (upper right penal) or ATRA + HFHFr diets (lower left panel). Insets: high magnification images of selected regions.
HEP_25798_sm_SuppFig8.tif2478KSupporting Fig. 8. RARa recruitment in the Lepr promoter region in response to ATRA. (A) Putative direct repeats (DRs) for the binding of RAR in the Lepr promoter region. The sites were predicted by the NHR-scan program. Each sequence is shown below. Numbers (bp) correspond to the position relative to the transcription start site (+1). The pairs of inverted arrows indicate the primers for ChIP assays. INT primers used as a negative control for the ChIP assay were located in Lepr intron 1. (B) The relative enrichment of chromatin containing RARa determined by ChIP. Nuclear extracts prepared from TLR3 cells treated with DMSO (open columns) or 5 μM ATRA (filled columns) for 12 h were subjected to ChIP using an anti-RARa antibody. The amount of each DR in coprecipitated genomic DNA fragments was determined by qPCR. Mouse cytochrome P450 26a1 retinoic acid response element (CYP26A1-RARE) and INT were used as positive and negative controls, respectively. Values relative to input and normalized to those of INT are shown.
HEP_25798_sm_SuppFig9.tif3684KSupporting Fig. 9. Effects of 4-week Am80 treatment on body weight changes, hepatic steatosis, and insulin sensitivity of KK-Ay mice. (A) Body weight changes of KK-Ay mice from 0 to 4 weeks. (B) Overall average daily consumptions for 4 weeks feeding of experimental diets. (C-E) Hepatic non-esterified free fatty acid (NEFA) (C), triglyceride (TG) (D), and total cholesterol (E) contents in KK-Ay mice after 4 weeks feeding of experimental diets. (F) Homeostatic model assessment of insulin resistance (HOMA-IR) indices of KK-Ay mice fed the Am80-supplemented normal diet for 4 weeks. (G) Serum leptin levels in KK-Ay mice after 4 weeks feeding of the experimental diets. *P < 0.05; **P < 0.01 between Am80 diet (filled columns) vs. control diet (open columns).
HEP_25798_sm_SuppTab1and2.doc111KSupporting Information Table 1 and 2.
HEP_25798_sm_SuppInfo.doc68KSupporting Information

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