Research ArticleLiver disease

Inhibition of ileal bile acid uptake protects against nonalcoholic fatty liver disease in high-fat diet–fed mice

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Science Translational Medicine  21 Sep 2016:
Vol. 8, Issue 357, pp. 357ra122
DOI: 10.1126/scitranslmed.aaf4823
  • Fig. 1. Administration of an ASBTi increases fecal BA excretion and hepatic BA synthesis.

    (A) Study design showing diets and durations of ASBTi (SC-435) treatments: chow (n = 12), HFD (n = 12), HFD/ASBTi16w (n = 16), and HFD/ASBTi4w (n = 8). (B) Fecal BA excretion in HFD and HFD/ASBTi16w feeding groups. (C) Hepatic expression of Cyp7a1 and Cyp8b1 mRNA. (D and E) Ileal Fgf15 mRNA expression (D) and colonic Ibabp mRNA expression (E). The labeling scheme for each group is indicated in the embedded legend. Means + SD are shown. Distinct lowercase letters indicate significant differences between groups; individual P values are provided in table S1.

  • Fig. 2. Administration of an ASBTi restores glucose tolerance.

    (A) Body weight gain in the indicated ad libitum–fed groups. * indicates value significantly different from WT. (B) Final body weight. (C) Average caloric intake over 16 weeks of ad libitum feeding. (D) Liver weight to body weight ratio after 16 weeks. (E) Glucose tolerance tests (GTTs). * indicates values significantly different between chow and HFD; # indicates values significantly different between chow and HFD/ASBTi4w. Individual P values are provided in table S1. (F) GTT area under the curve (AUC) (gram per liter per minute). The labeling scheme for each group is indicated in the embedded legend. Means + SD are shown. Distinct lowercase letters indicate significant differences between groups).

  • Fig. 3. Administration of an ASBTi reduces hepatic steatosis and accumulation of TGs and cholesterol.

    (A to D) Hematoxylin and eosin–stained liver sections from (A) chow-, (B) HFD-, (C) HFD/ASBTi16w-, and (D) HFD/ASBTi4w-fed mice. (E) NAS. (F) Steatosis score. (G) Hepatic TG content. (H) Hepatic cholesteryl ester content. The labeling scheme for each group is indicated in the embedded legend. Means + SD are shown. Different lowercase letters indicate significant differences between groups; individual P values are provided in table S1. Scale bars, 100 μm.

  • Fig. 4. Administration of an ASBTi shifts hepatic BA composition toward FXR agonism.

    (A and B) Total hepatic BA content (A) and composition (B) (TMCA, tauromuricholic acid; MCA, muricholic acid, THDCA, taurohyodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; TLCA, taurolithocholic acid; CA, cholic acid). (C) Hydrophobicity index. (D) Pie charts for hepatic FXR antagonist/non-agonist (black) and agonist (white) BA content. (E) βTMCA inhibits TCA-induced FXR activation in HepG2 cells. (F) βTMCA inhibits TCDCA-induced FXR activation in HepG2 cells. The concentration of the individual BAs is indicated on the x axis. (G) Activation of an FXR reporter plasmid (pECRELuc) in transfected HepG2 cells by composite mixtures of the six major BAs (3 μM final concentration) modeling the in vivo hepatic BA content from the indicated groups. The vehicle (Veh) used was methanol, and positive control consisted of 3 μM TCDCA. The composition of the BA mixtures used in these in vitro studies is shown in table S3. The labeling scheme for each group is indicated in the embedded legend. Means + SD are shown. Distinct lowercase letters indicate significant differences between groups; individual P values are provided in table S1.

  • Fig. 5. Inhibition of ileal ASBT function alters global hepatic gene expression.

    (A) Venn diagram of RNA-seq analysis of the two comparisons: HFD versus chow and HFD versus HFD/ASBTi16w. (B) Heat map comparing liver gene expression in HFD versus HFD/ASBTi16w groups. (C) Ontology analysis/pathway (Panther) analysis of HFD versus HFD/ASBTi16w as down-regulated (white bars) and up-regulated (black bars) pathways.

  • Fig. 6. Administration of an ASBTi alters hepatic and intestinal gene expression.

    (A) Hepatic mRNA expression of genes involved in BA signaling and transport, cholesterol synthesis, lipid synthesis, lipid droplet formation, fatty acid oxidation, inflammation, and fibrosis. (B and C) mRNA expression of genes involved in BA signaling and transport in the ileum (B) and (C) colon. Quantitative real-time PCR was performed on chow, HFD, HFD/ASBTi16w, and HFD/ASBTi4w groups, and expression shown relative to chow (set as 1). For clarity, gene expression in the HFD and HFD/ASBTi16w groups are displayed. Cyclophilin was used as a housekeeping gene to normalize mRNA expression. The labeling scheme for each group is indicated in the embedded legend. Means + SD are shown. Distinct lowercase letters indicate significant differences between groups; individual P values are provided in table S1.

  • Fig. 7. Hepatic accumulation of TGs and cholesterol is reduced in Asbt−/− mice.

    (A) Body weight gain in the indicated ad libitum–fed groups. * indicates value significantly different from WT. (B) Average caloric intake over 1 week of feeding. (C) Liver weight after 1 week of HFD or chow. (D) Liver weight to body weight ratio after 1 week of HFD or chow. (E to H) Hematoxylin and eosin–stained liver sections from (E) WT/chow, (F) WT/HFD 1 week, (G) Asbt−/−/chow, and (H) Asbt−/−/HFD 1 week mice. (I) Hepatic TG content. (J) Hepatic total cholesterol content. (K) Hepatic free cholesterol content. (L) Hepatic cholesteryl ester content. The labeling scheme for each group is indicated in the embedded legend. Means + SD are shown. Distinct lowercase letters indicate significant differences between groups; individual P values are provided in table S1. Scale bars, 100 μm.

  • Fig. 8. Hepatic BA composition is altered in Asbt−/− mice.

    (A) Total hepatic BA content and (B) BA composition. (C and D) Hydrophobicity index (C) and pie charts (D) for hepatic FXR antagonist/non-agonist (black) and agonist (white) BA content. (E) Hepatic gene expression in WT and Asbt−/− mice fed a HFD for 1 week. Values shown are relative to chow-fed WT mice. (F) Protein expression of full-length (fl) SREBP1 increased in HFD-fed WT mice but not in HFD-fed Asbt−/− mice. SREBP1 expression was normalized to the amount of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the quantitation is shown below the blot. The labeling scheme for each group is indicated in the embedded legend. Means + SD are shown. Distinct lowercase letters indicate significant differences between groups; individual P values are provided in table S1.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/357/357ra122/DC1

    Materials and Methods

    Fig. S1. Inhibition of ileal BA absorption does not affect intestinal and colonic gene expression, food and water consumption, and hepatic injury markers.

    Fig. S2. Inhibition of ileal BA absorption improves whole-body insulin tolerance and hepatic cholesterol concentration.

    Fig. S3. Inhibition of ileal BA absorption does not affect hepatic fibrosis.

    Fig. S4. Inhibition of ileal BA absorption changed hepatic BA composition and correlated with TG and cholesterol concentrations.

    Fig. S5. Inhibition of ileal BA absorption changed global hepatic gene expression during 16 weeks of HFD feeding.

    Fig. S6. Inhibition of ileal BA absorption changed gene expression in the mouse liver.

    Fig. S7. Inhibition of ileal BA absorption does not affect hepatic and ileal ceramide amount and composition.

    Fig. S8. Inhibition of ileal BA absorption affects hepatic gene expression in WT and Asbt−/− mice fed with chow or HFD for 1 week.

    Table S1. P values for all comparisons (provided as an Excel file).

    Table S2. Individual elements of NAS.

    Table S3. Composite BA mixtures of six BAs representing the major in vivo hepatic BA species in HFD and HFD/ASBTi16w mice.

    Table S4. Differentially expressed genes and pathway analysis (provided as an Excel file).

    Table S5. HFD composition and macronutrient information.

    Table S6. Primer sequences used for real-time PCR analysis.

    Table S7. Primary data (provided as an Excel file).

    References (4253)

  • Supplementary Material for:

    Inhibition of ileal bile acid uptake protects against nonalcoholic fatty liver disease in high-fat diet–fed mice

    Anuradha Rao, Astrid Kosters, Jamie E. Mells, Wujuan Zhang, Kenneth D. R. Setchell, Angelica M. Amanso, Grace M. Wynn, Tianlei Xu, Brad T. Keller, Hong Yin, Sophia Banton, Dean P. Jones, Hao Wu, Paul A. Dawson, Saul J. Karpen*

    *Corresponding author. Email: skarpen{at}emory.edu

    Published 21 September 2016, Sci. Transl. Med. 8, 357ra122 (2016)
    DOI: 10.1126/scitranslmed.aaf4823

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Inhibition of ileal BA absorption does not affect intestinal and colonic gene expression, food and water consumption, and hepatic injury markers.
    • Fig. S2. Inhibition of ileal BA absorption improves whole-body insulin tolerance and hepatic cholesterol concentration.
    • Fig. S3. Inhibition of ileal BA absorption does not affect hepatic fibrosis.
    • Fig. S4. Inhibition of ileal BA absorption changed hepatic BA composition and correlated with TG and cholesterol concentrations.
    • Fig. S5. Inhibition of ileal BA absorption changed global hepatic gene expression during 16 weeks of HFD feeding.
    • Fig. S6. Inhibition of ileal BA absorption changed gene expression in the mouse liver.
    • Fig. S7. Inhibition of ileal BA absorption does not affect hepatic and ileal ceramide amount and composition.
    • Fig. S8. Inhibition of ileal BA absorption affects hepatic gene expression in WT and Asbt−/− mice fed with chow or HFD for 1 week.
    • Legend for table S1
    • Table S2. Individual elements of NAS.
    • Table S3. Composite BA mixtures of six BAs representing the major in vivo hepatic BA species in HFD and HFD/ASBTi16w mice.
    • Legend for table S4
    • Table S5. HFD composition and macronutrient information.
    • Table S6. Primer sequences used for real-time PCR analysis.
    • Legend for table S7
    • References (4253)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. P values for all comparisons (provided as an Excel file).
    • Table S4. Differentially expressed genes and pathway analysis (provided as an Excel file).
    • Table S7. Primary data (provided as an Excel file).

    [Download Tables S1, S4, and S7]

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