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Hepatic Gene Expression of Bile Acid Synthesis Genes from Wild-Type and Fxr−/− Mice

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Hepatic Gene Expression of Bile Acid Synthesis Genes from Wild-Type and Fxr−/− Mice A 2.0 Acox2 B 2.0 Akr1c14 C 2.0 Akr1d1 D 2.0 Amacr ** 1.5 1.5 1.5 1.5 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA 0.0 0.0 0.0 0.0 GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− E F 2.0 Cyp7b1 2.0 Cyp27a1 G 2.0 Cyp39a1 H 2.0 Hsd3b7 1.5 1.5 1.5 1.5 * 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA 0.0 0.0 0.0 0.0 GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− I J K 2.0 Hsd17b4 2.0 Scp2 2.0 Slc27a5 L Fxr Cyp7a1 Cyp8b1 1.0 1.5 1.5 1.5 * 1.0 1.0 1.0 0.5 *** *** 0.5 0.5 0.5 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA *** *** *** 0.0 0.0 0.0 0.0 *** GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h GSK − 30’ 1h 2h − 30’ 1h 2h Time (h) 0 4 16 0 4 16 0 4 16 FXR WT Fxr−/− FXR WT Fxr−/− FXR WT Fxr−/− post plating M N CYP7A1 CYP8B1 Supplementary Figure 1 – FXR activation leads to rapid changes in gene expression 1.0 1.0 (A-K) Hepatic gene expression of bile acid synthesis genes from wild-type and Fxr−/− mice. Mice were treated with either vehicle (WT n=16; Fxr−/− n=7) or GSK2324 and RNA isolated after 30 minutes (WT n=12; Fxr−/− n=6), 1h (WT n=10; Fxr−/− n=6) or 2h (WT n=8; Fxr−/− 0.5 0.5 mRNA half-life n=7). Gene expression analysis was determined by RT-qPCR and normalized to Tbp. (L) mRNA (Fold Change) mRNA mRNA half-life mRNA (Fold Change) mRNA Gene expression of Cyp7a1, Cyp8b1, and Fxr from primary hepatocytes that were freshly 0.0 0.0 isolated (t=0) or the same hepatocytes plated for 4 or 16 hours on collagen coated plates 012345678 012345678 (n=6 wells/condition). (M,N) CYP7A1 and CYP8B1 mRNA levels in IHH cells treated with Time (h) Time (h) actinomycin D for various time points. Gene expression was determined by RT-qPCR and data are normalized to 36B4. Data are normalized to 36B4. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; *** p<0.001) by one way ANOVA. A 2.0 Cyp8b1 (Liver) D 1.5 Cyp8b1 (Liver) 1.5 1.0 * ** 1.0 * 0.5 0.5 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA ** 0.0 0.0 GSK − 30’ 1h 2h 4h − 30’ 1h 2h 4h GSK − 30’ 1h 2h FXR Fxrflox-flox FxrL-KO Shp−/− 4 B 20 Shp (Liver) E Insig2a (Liver) *** *** ** 3 *** 15 *** *** 10 2 5 1 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA 0 0 GSK − 30’ 1h 2h 4h − 30’ 1h 2h 4h GSK − 30’ 1h 2h FXR Fxrflox-flox FxrL-KO Shp−/− C 4 Fabp6/iBabp (Ileum) 3 *** *** *** 2 ** * * 1 mRNA (Fold Change) mRNA 0 GSK − 30’ 1h 2h 4h − 30’ 1h 2h 4h FXR Fxrflox-flox FxrL-KO Supplementary Figure 2 – Decrease in Cyp8b1 following FXR activation required hepatic FXR but is independent of SHP (A-C) Gene expression of hepatic Cyp8b1 and Shp and ileal Fabp6 in littermate Fxrflox-flox and FxrL-KO mice were treated with either vehicle (n=13 Fxrflox-flox; n=11 FxrL-KO) or GSK2324 for 30 minutes (n=9 Fxrflox-flox; n=8 FxrL-KO), 1h (n=7 Fxrflox-flox; n=9 FxrL-KO), 2h (n=8 Fxrflox-flox; n=7 FxrL-KO) or 4h (n=5 Fxrflox-flox; n=10 FxrL-KO). (D- E) Gene expression of hepatic Cyp8b1 and Insig2a in Shp−/− mice treated with either vehicle (n=8) or GSK2324 for 30 minutes (n=8), 1h (n=7) or 2h (n=8). Gene expression analysis was determined by RT-qPCR and normalized to Tbp. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; *** p<0.001) by one way ANOVA. A B C CDCA Veh GW D Cyp7a1 Cyp8b1 Shp I.B. 1.5 4 ZFP36L1 1.5 CYP7A1 *** I.B. PDI 3 1.0 2.0 ZFP36L1 1.0Legend * Legend ** fold fold change) fold fold change) 2 1.5 ( ( Legend 0.5 1.0 1 fold change) 0.5 ( mRNA mRNA mRNA mRNA *** *** *** 0.5 *** 0.0 0 (Fold Change) mRNA mRNA mRNA Diet Ctr CA Ctr CA Diet Ctr CA 0.0 0.0 Diet Diet Diet Treatment CDCA Veh GW4064 Treat. CDCA Veh GW4064 E F G 4 4 ZFP36L1 ZFP36L1 ZFP36L1 Vehicle 1.00 30' 3 3 ** 1h 0.75 *** ** 2 2h * 2 * 0.50 ** 4h 1 0.25 1 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA 0.00 0 02468 0 Time − 30’ 1h 2h Time − 30’ 1h 2h Time (h) Treat. GW4064 (1µM) Treat. CDCA (100µM) Supplementary Figure 3 – Zfp36l1 is a direct FXR target gene (A,B) Hepatic Cyp7a1,Cyp8b1 and Shp mRNA expression in C57BL/6 wild-type fed a control diet (Ctr; n=8) or a diet supplemented with 0.5% cholic acid (CA Diet; n=8) for 7 days. Gene expression analysis was determined by RT-qPCR and normalized to Tbp. (C) ZFP36L1 mRNA and protein (D) CYP7A1 mRNA in immortalized human hepatocytes (IHH) treated with either CDCA (100µM) or GW4064 (1µM) for 24h (n=3 wells/condition). (E) ZFP36L1 mRNA levels were measured in IHH cells treated with actinomycin D for the indicated times (n=6 wells condition). (F and G) ZFP36L1 mRNA levels in IHH cells treated with either CDCA (100µM) or GW4064 (1µM) for 30’, 1h and 2h (n=4 wells/condition). Gene expression data was normalized to 36B4 or TBP. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; *** p<0.001) by Student’s t-test for A-D and one-way ANOVA for F-G. A I.B. ZFP36L1 I.B. PDI B C 4 1.5 1.5 Zfp36l1 Cyp7a1 Ad-Ctr Ad-Zfp36l1 3 *** 1.0 1.0 2 ** ** ** 0.5 0.5 1 mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA mRNA (Fold Change) mRNA 0 0.0 0.0 Scp2 Acox2 Akr1d1 Amacr Cyp8b1 Cyp7b1 Hsd3b7 Akr1c14 Cyp27a1Cyp39a1 Hsd17b4 Slc27a1 D E F G H I ) 4000 15 1.5 250 25 40 l) M Insulin Glucose ALT AST µ µ ) 200 20 3000 dL 30 * 10 1.0 150 15 2000 20 100 10 ALT (U/L) ALT 5 0.5 (U/L) AST 1000 Insulin (ng/ml) 10 Glucose (mg/ 50 5 Gall Bladder Volume ( Gall Bladder Volume 0 0 0.0 0 0 0 Biliary BAs/gall bladder ( bladder BAs/gall Biliary Supplementary Figure 4 – ZFP36L1 gain-of-function decreases CYP7A1 and alters bile acids in vivo (A) Zfp36l1 mRNA and protein and (B) Cyp7a1 and (C) other bile acid synthesis genes in female C57BL/6 wild-type mice treated with either Ad-control (Ad-Ctr) or Ad-Zfp36l1 for 5 days (n=6-7 mice/group). (D) Bile acid amount and (E) gall bladder volume (F) plasma insulin (G) plasma glucose (H) plasma alanine aminotransferase (ALT) and (I) plasma aspartate aminotransferase (AST) in male Ad-control (Ad-Ctr) or Ad-Zfp36l1 treated mice (n=10 mice/group). Gene expression analysis was determined by RT-qPCR and normalized to Tbp. All data shown as mean ± SEM. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001) by Student’s t-test. I VO2 (ml/kg/hr) A with repeated measures for I for measures repeated with ** (*p<0.05; differences All mice VO in glucose plasma (D) Biliary (A) 5 Figure Supplementary 2000 4000 6000 8000 Biliary BAs/gall bladder 10000 15000 0 (µM) 5000 7am data shown as mean mean as shown data 2 plasma plasma 8am , (J) VCO (J) , 9am 0 Zfp36l1 Zfp36l1 (n=6) placed in metabolic cages at 60 days of age fed a standard rodent diet. diet. rodent standard a fed age of days 60 at cages metabolic in placed (n=6) 10am 11am 12pm 1pm aspartate aspartate 2pm 3pm fl L - - 4pm fl KO bile acid amount amount acid bile 5pm littermate male male littermate 6pm free fatty acids (FFA) (E) plasma plasma (E) (FFA) acids fatty free 7pm ** 8pm 2 9pm 10pm B (K) Activity of littermate littermate Activityof (K) 11pm 12am Gall Bladder Volume 1am 2am (µl) 10 20 30 40 aminotransferase (AST) (G) plasma insulin and (H) plasma plasma (H) and insulin plasma (G) (AST) aminotransferase 3am 4am 0 5am 6am 7am 8am 9am p<0.01 10am 11am ± 12pm 1pm Zfp36l1 – * SEM. Asterisks indicate statistically significant significant statistically indicate Asterisks SEM. 2pm (B) 3pm ZFP36L1 4pm - 5pm K.
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