Supplementary Materials 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@emory.edu

Published 21 September 2016, Sci. Transl. Med. 8, 357ra122 (2016)

DOI: 10.1126/scitranslmed.aaf4823

The 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.

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

Legend for table S7

References (42–53)

Other Supplementary Material for this manuscript includes the following:

(available at

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

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).

Materials and Methods:

Study Design

The experiments presented in this manuscript were designed to test the overall hypothesis that

inhibition of ileal ASBT improved molecular and histologic aspects of NAFLD in mice. There is

conflicting evidence in the literature regarding the benefits of inhibiting or activating ileal FXR

signaling as a means to improve features of NASH. Our strategy employed both pharmacologic

and genetic inhibition of ASBT function in the setting of mice fed the ALIOS high fat diet. For

uniformity and reproducibility, male mice for the ASBTi studies were obtained from one source,

used at the same ages, treated with the same diets, and housed in one facility. Experiments

were repeated at least three times, and analyses conducted according to standard lab practices.

Histology was analyzed in a blinded fashion, as were all statistical analyses. In vitro treatment of

HepG2 cells with synthetic BA mixtures of the 6 most prevalent conjugated BA species

designed to mimic BA composition in vivo was performed after liver BA analyses.

Animals and experimental design

The Emory University Institutional Animal Care and Use Committee approved all experiments.

Male C57Bl/6J mice were obtained from Jackson Laboratories. The Asbt-/- mice have been

previously described (42) and a colony is maintained at Emory University School of Medicine.

Animals were group housed in laboratory cages at 23° C under a 12-hour light/dark cycle.

Starting at four to five weeks of age, mice were fed ad libitum a standard chow, a high fat diet

which included 0.2% (w/w) cholesterol (HFD) (21, 43) (Harlan Teklad custom diet TD.120330),

or the HFD plus 0.006% of an ASBTi (SC-435) (HFD/ASBTi16w) (44), providing approximately

11 mg/kg/day. The HFD derives 45% of calories from fat in the form of partially hydrogenated

soybean oil (fatty acid composition: 28% saturated, 57% monounsaturated, 13%

polyunsaturated; trans-fat custom diet TD06303, Harlan Teklad) (table S5). The HFD-fed mice

also received ad libitum drinking water containing 42 g per liter of a mixture of fructose (55%

w/w) and glucose (45% w/w). An additional group of mice was fed the HFD for 12 weeks, and

then switched to the HFD plus ASBTi for 4 weeks (HFD/ASBTi4w). A graphical representation

of the study design and treatment groups is shown in fig. 1A. Each group included 8 to 16 mice.

Food and water consumption was measured by weighing new and remaining food and water

two times weekly. Calorie intake was calculated by multiplying the weight of the food and sugar

water consumed by their caloric density. Body weight was measured weekly. Glucose and

insulin tolerance tests were performed at week 15. At the end of the study, fecal samples and

fasting (6 hours) blood samples were obtained. At necropsy, the liver was collected, and

portions were formalin-fixed or snap-frozen in liquid nitrogen and stored at -80° C for

subsequent analysis. Small intestines were collected, snap-frozen in liquid nitrogen, and stored

at -80°C. Male C57Bl/6J wild type and Asbt-/- mice (13-14 weeks; n=10-13) were maintained on

chow or fed ad libitum with the HFD and ad libitum drinking water containing 42 g per liter of a

mixture of fructose (55% w/w) and glucose (45% w/w) for one week, as described above. Food

and water consumption was monitored. After one week, the mice were sacrificed, and liver and

intestinal tissues were collected for analysis.

Glucose and insulin tolerance testing

Glucose and insulin testing was performed as described (43). Briefly, for the glucose tolerance

test, mice were fasted for 6 hours, and glucose (2 g/kg) was administered intraperitoneally using

a 31-gauge insulin syringe. Glucose concentrations were measured at 0, 15, 30, 60, 90, and

120 min by tail vein sampling with a portable glucometer. For insulin tolerance, mice were fasted

for 4 hours and then injected intraperitoneally with human regular insulin (0.1 U/kg). Glucose

concentrations were measured by tail vein sampling using a portable glucometer at 0, 15, 30,

45, and 60 min.

Plasma biochemistries and liver lipids

A fasting blood sample (6 hour fast) was collected by cardiac puncture at the end of the study to

measure plasma chemistries, including total protein, albumin, alkaline phosphatase, alanine

aminotransferase, aspartate aminotransferase, and cholesterol. Plasma chemistries were

measured at the University of Georgia Veterinary Diagnostic Laboratories. Hepatic

concentrations of total cholesterol (Pointe Scientific), free cholesterol, and triglyceride (Wako

Diagnostics) were measured by enzymatic assays (42).

Histology

The livers were removed and weighed, and a portion was formalin-fixed, embedded in paraffin,

and stained with hematoxylin and eosin. Sirius red staining was performed on paraffin-

embedded liver sections (method adapted from Picrosirius Red Stain Kit, Polysciences, Inc.).

The liver histology was blindly assessed by H.Y. for steatosis, lobular inflammation, and

hepatocellular ballooning to derive the NAS; the extent of fibrosis was assessed on a 0 to +4

scale as described (45). Hepatic hydroxyproline content was measured using a colorimetric

assay as described (46).

BA and ceramide analysis

For fecal BA measurements, feces were collected and extracted for quantitation of total BA by

enzymatic assay (22). Quantitative analysis of tissue BA and ceramide was carried out using a

Waters Quattro Premier XE triple quadruple mass spectrometer interfaced with Aquity UPLC

system. Mouse liver was homogenized and sonicated sequentially in 80% methanol/water and

chloroform/methanol (2:1, v/v). After centrifugation, the supernatants were combined and dried.

For LC-MS analysis of ceramides, the dried extracts were resuspended in methanol containing

internal standard. For liver BA analysis, the samples underwent further solid-phase extraction

before analysis. Quantification of BAs was based on a validated isotope dilution mass

spectrometry method (47). Calibration curves were built with 20 of the mouse BAs using the

single ion recording (SIR) function on the mass spectrometer. Ceramides were quantified using

3 ceramide reference standards (C16, C18, and C24 ceramide) based on the multiple reaction

monitoring (MRM) function on mass spectrometer. Total BAs or total ceramides were calculated

from the sum of individual BA or ceramide species, respectively, that were detected in mouse

liver, and their amounts were normalized to tissue weight.

RNA-seq analysis

Total hepatic RNA was isolated using miRNAeasy kit (Qiagen) according to the manufacturer’s

instructions. RNA-seq libraries were prepared with the Illumina TruSeq NA preparation kit and

sequenced on an Illumina HiSeq1000 system. Sequences were aligned to the mouse genome

(NCBI37/mm9, July 2007) using Bowtie 0.12.7 (48) with options: "bowtie -v 3 -q -S”. Reads

mapped to the bodies of RefSeq genes were obtained using Bioconductor (49). The number of

reads that mapped to each gene was used to quantify RNA expression. Pairwise comparisons

between three groups were performed to detect differentially expressed (DE) genes using the

Bioconductor package DSS (50). Differentially expressed genes with a fold change > 1.5 and

P<0.05 were selected for functional annotation. Ontology and pathway analysis was performed

using DAVID (51).

RNA and protein expression

RNA isolation and real time PCR were performed as described previously (52, 53). The primer

sequences are shown in table S6 and were obtained from literature or designed using Primer 3

and Roche primer design. Western blot analysis was performed as described previously (52,

53).

In vitro transfection and luciferase experiments

HepG2 cells were obtained from American Type Culture Collection. Cells were grown as

monolayers at 37°C in a humidified atmosphere of 95% air and 5% CO2 in medium that

consisted of modified Eagle's minimal essential medium (EMEM) supplemented with 10% fetal

bovine serum (FBS), 1% penicillin-streptomycin, and 1% l-glutamine (GIBCO). For transient

transfections, HepG2 cells were plated in 12-well plates (105 cells/well). After 24 hours, cells

were transfected with Lipofectamine3000 (Invitrogen) transfection reagent. The luciferase

reporter plasmid pECRE-Luc (0.5 μg) was transfected together with pRSVrenilla (0.01 μg) to

monitor transfection efficiency. In addition, cells were co-transfected with human FXR (FXRα1

isoform; also called FXRα1(+), which initiates in exon 1 of the FXR gene and includes the 12-bp

insert in exon 5 resulting from alternative splicing; 0.05 μg), human RXRα (0.05 μg), and human

NTCP (0.5 μg) expression plasmids. Next day, the cells were treated with different

concentrations of BAs (TCA, TDCA, TCDCA, αTMCA, βTMCA, ωTMCA) in serum-free medium.

After 24 hours, cells were harvested and processed using Luciferase assay reagents (Dual-

Luciferase Reporter Assay System) from Promega, and the bioluminescence was monitored on

a luminometer (Synergy HTX, Biotek). The results were normalized to RSVrenilla activity and

represented as relative fold change.

Statistical analysis

Mean values ± SD are shown unless otherwise indicated. The data were evaluated using

GraphPad Prism software, assessing for statistically significant differences using the two-tailed

Student’s t test or ANOVA with the Tukey-Kramer honestly significant difference test for post-

hoc analysis. Correlations were performed using a Spearman Test. Differences were

considered statistically significant at a P value <0.05 and are indicated by different lowercase

letters in the figures. Primary data are provided in table S7 and exact P values are provided in

table S1.

Fig. S1. Inhibition of ileal BA absorption does not affect intestinal and colonic gene

expression, food and water consumption, and hepatic injury markers. (A) Relative mRNA

expression of Asbt in ileum. (B) Relative mRNA expression of Asbt in colon. (C) Relative mRNA

expression of Akr1b7 in colon. (D) Colonic expression of genes involved in ER stress. (E)

Colonic expression of oncogenic signaling genes. (F) Colonic expression of genes involved in

innate immunity and inflammation. (G) Food and (H) water consumption. (I) Serum chemistry

markers. The labeling scheme for each group is indicated in embedded legend. Mean + SD are

shown. Distinct lowercase letters indicate significant differences between groups; individual P

values are provided in table S1.

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

hepatic cholesterol concentration. (A) Insulin tolerance and (B) AUC. (C) Hepatic total

cholesterol concentration. (D) Hepatic free cholesterol concentration. The labeling scheme for

each group is indicated in embedded legend. Mean + SD are shown. Distinct lowercase letters

indicate significant differences between groups; individual P values are provided in table S1. In

(A), * indicates different from chow for HFD, ¶ indicates different from chow for HFD/ASBTi16w,

# indicates different from chow for HFD/ASBTi4w.

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

for livers from (A) chow, (B) HFD, (C) HFD/ASBTi16w, and (D) HFD/ASBTi4w mice. (E)

Quantification of Sirius red staining. (F) Biochemical analysis of fibrosis via quantification of

hydroxyproline. Scale bars in A-D indicate 100 µm. The labeling scheme for each group is

indicated in embedded legend. Mean + SD are shown. Distinct lowercase letters indicate

significant differences between groups; individual P values are provided in table S1.

Fig. S4. Inhibition of ileal BA absorption changed hepatic BA composition and correlated

with TG and cholesterol concentrations. Correlation of hepatic TG and cholesterol content

with hepatic muricholic acid content (A-B) and (C-D) hepatic FXR agonistic BA (TCDCA+TDCA)

content, respectively. The labeling scheme for each group is indicated in the embedded legend.

Fig S5. Inhibition of ileal BA absorption changed global hepatic gene expression during

16 weeks of HFD feeding. (A) Venn diagram of the differentially expressed genes in each

treatment group based on RNA-seq analysis (B) Heat maps comparing liver gene expression in

HFD vs chow groups (C) Pathway analysis of HFD versus chow.

Fig S6. Inhibition of ileal BA absorption changed gene expression in mouse liver. (A)

mRNA expression of genes involved in BA signaling and transport in liver, (B) ileum, and (C)

colon. (D) mRNA expression of genes involved in hepatic cholesterol and lipid biosynthesis, lipid

transport, and fatty acid oxidation. (E) mRNA expression of genes involved in hepatic lipid

droplet formation, inflammation, and fibrosis. The labeling scheme for each group is indicated in

the embedded legend. Mean + SD are shown. Distinct lowercase letters indicate significant

differences between groups; individual P values are provided in table S1.

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

amount and composition. (A) Total and (B) individual ceramide species concentrations in the

liver. (C) Total and (D) individual ceramide species concentrations in the ileum. The labeling

scheme for each group is indicated in embedded legend. Mean + SD are shown. Distinct

lowercase letters indicate significant differences between groups; individual P values are

provided in table S1.

Fig. S8. Inhibition of ileal BA absorption affects hepatic gene expression in WT and

Asbt–/– mice fed chow or a HFD for 1 week. Values shown are relative to chow-fed WT mice.

The labeling scheme for each group is indicated in embedded legend. Mean + SD are shown.

Distinct lowercase letters indicate significant differences between groups; individual P values

are provided in table S1.

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

Table S2: Individual elements of NAS.

Diet Steatosis

Lobular

Inflammation

Hepatocyte

ballooning

Chow 0 (0)a 0.67 (0.49) 0 (0)a

HFD 3 (0)b 1.06 (0.68) 0.69 (0.48)b

HFD/ASBTi16wk 1.92 (0.76)c 0.85 (0.38) 0.08 (0.28)a

HFD/ASBTi4wk 2.75 (0.46)b 0.87 (0.35) 0.75 (0.46)b

NAS determined based on (27). Distinct lowercase letters indicate significant

differences between groups. Individual P values are provided in table S1.

Table S3: Composite BA mixtures of six BAs representing the major in vivo

hepatic BA species in HFD and HFD/ASBTi16w mice.

BA HFD mix (% BA) HFD/ASBTi16w mix (%BA)

ωTMCA 14.0 1.0

αTMCA 14.2 7.8

ßTMCA 28.7 4.7

TCA 38.0 48.7

TCDCA 3.4 10.1

TDCA 1.6 27.6

Table S4: Differentially expressed genes and pathway analysis (provided as an

Excel file).

Table S5: HFD composition and macronutrient information.

Component g/Kg

Casein 230.0

DL-Methionine 3.4

Sucrose 211.5

Corn starch 80.0

Maltodextrin 140.0

Hydrogenated vegetable oil (Primex) 220.0

Soybean oil 10.0

Cholesterol 2.0

Cellulose 50.0

Mineral mix, AIN-93G-MX 46.0

Calcium Phosphate, dibasic 3.3

Niacin 0.0420

Calcium pantothenate 0.0224

Pyridoxine HCl 0.0098

Thiamin HCl 0.0084

Riboflavin 0.0084

Folic Acid 0.0028

Biotin 0.0003

Vitamin B12 (0.1% in mannitol) 0.0350

Vitamin E, DL-alpha tocopheryl acetate (500 IU/g) 0.1000

Vitamin A Palmitate (500,000 IU/g) 0.0112

Vitamin D3, cholecalciferol (500,000 IU/g) 0.0028

Vitamin K1, phylloquinone 0.0011

Choline bitartrate 3.3000

TBHQ, antioxidant 0.0460

% kcal from Chow HFD

Protein 24 17.7

Carbohydrate 60 37

Fat 16 45.3

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

Primer sets used for HFD-ASBTi- experiments

Gene name Forward primer 5'-3' Reverse primer 5'-3'

Cyclophilin GGCCGATGACGAGCCC TGTCTTTGGAACTTTGTCTGCA

Cyp7a1 CAGGGAGATGCTCTGTGTTCA AGGCATACATGCAAAACCTCC

Cyp8b1 TTCGACTTCAAGCTGGTCGA CAAAGCCCCAGCGCCT

Cyp7b1 AATTGGACAGCTTGGTCTGCCT TGTGTATGAGTGGAGGAAAGAGGG

Cyp27a1 GCCTTGCACAAGGAAGTGACT CGCAGGGTCTCCTTAATCACA

Fxr TCCACAACCAAGTTTTGCAG TCTCTGTTTGTTGTACGAATCCA

Shp AAGGGCACGATCCTCTTCAA CTGTTGCAGGTGTGCGATGT

Abcb11 CTGCCAAGGATGCTAATGCA CGATGGCTACCCTTTGCTTC

Ostβ GATGCGGCTCCTTGGAATTA GGAGGAACATGCTTGTCATGAC

Tgr5 GTCAGCTCCCTGTTCTTTGC CAGGAGGCCATAAACTTCCA

S1pr2 GCGTGGTCACCATCTTCTCC CGTCTGAGGACCAGCAACATC

Sphk2 AGACGGGCTGCTTTACGAG CAGGGGAGGACACCAATG

Asbt TGGGTTTCTTCCTGGCTAGACT TGTTCTGCATTCCAGTTTCCAA

Ibabp CAAGGCTACCGTGAAGATGGA CCCACGACCTCCGAAGTCT

Fgf15 GAGGACCAAAACGAACGAAATT ACGTCCTTGATGGCAATCG

Gcg CCAGTGATGTGAGTTCTTACTTGG CAATGG CGACTTCTTCTGG

Hsd3b5 GCTCTTGGAAACCACAAGGAAC GACAATCCTCTGGCCAAGAAAC

Hsd3b7 CGCTTTGGAGGTCGTCTATT CAGTATGTGCATCCAAGCAAC

Cyp3a11 GGATGAGATCGAT GAGGCTCTG CAGGTATTCCATCT CCATCACAGT

Srebp2 GCGTTCTGGAGACCATGGA ACAAAGTTGCTCTGAAAACAAATCA

Hmgcr CCGGCAACAACAAGATCTGTG ATGTACAGGATGGCGATGCA

Ch25oh TGCTACAACGGTTCGGAGC AGAAGCCCACGTAAGTGATGAT

Lxra TGAGAGCATCACCTTCCTCA TGGAGAACTCAAAGATGGGG

Abcg5 AGAGTCAGGATGGCCTGTAT ATGCTGAGCAGGGCCACTAT

Abcg8 GAGAGCTTCACAGCCCACAA GCCTGAAGATGTCAGAGCGA

Srebp1 GGAGCCATGGATTGCACATT CCTGTCTCACCCCCAGCATA

Scd1 TGTCTCGGTGTGTGTCGGAGT TGTACCACTACCTGCCTGCATG

Pparg CACAATGCCATCAGGTTTGG GCTGGTCGATATCACTGGAGA

Acc1/Acaca GAGAGGGGTCAAGTCCTTCC CTGCTGCCGTCATAAGACAA

Fasn TTGCTGGCACTACAGAATGC AACAGCCTCAGAGCGACAAT

Fads1 ACCCACCAAGAATAAAGCGCTAA CAGCCACATCCAGCAGCAG

Fads2 ACCGTGGCAAAAGCTCTCAG GAGAGGATGAACCAGGCAAGGC

Elovl6 AGCAGAGGCGCAGAGAACACGTA ATAAAGGCAGCGTACAGCGCAGAA

Elovl3 GCCTCTCATCCTCTGGTCCT TGCCATAAACTTCCACATCCT

Cd36 TTCCAGCCAATGCCTTTGC TGGAGATTACTTTTTCAGTGCAG

Ldlr CCACAGAACTGCCAGGGCCG GAATTCATCAGGTCGGCAGGT

Vldlr GAGCCCCTGAAGGAATGCC CCTATAACTAGGTCTTTGCAGATATGG

Mttp GACCACCCTGGATCTCCATA AGCGTGGTGAAAGGGCTTAT

Fabp1 CCATGAACTTCTCCGGCAAGTACC CTTTGGGTCCATAGGTGATGGTGAG

Fabp5 GGAAGGAGAGCACGATAACAAGA GGTGGCATTGTTCATGACACA

Ppara CTGGCATTTGTTCCGGTTCT TATTCGGCTGAAGCTGGTGT

Acaa1b GATTCCTATGGGGATAACTTCG ATGGTTTTCTTGTCACCCTTGT

Fgf21 CTGGGGGTCTACCAAGCATA CACCCAGGATTTGAATGACC

Cidea TCCTCGGCTGTCTCAATG TGGCTGCTCTTCTGTATCG

Cideb TCTGTGATCATAAGCGGACA GCAGCAGCGAGGAAGTCCAA

Cidec/Fsp27 GACTTTATTGGCTGCCTGAACG ATCTCCTTCACGATGCGCTT

Pnpla3 CGGGGCTACGCTATGTCTGAGC CCGCACGAGGTCCATGAGGATC

Pnpla2 ACGCCACTCACATCTACGGA CAATCAGCAGGCAGGGTCTT

Tm6sf2 CCTCGGTGGTGGACCTTGT TCCTTGGTGTAGAAATCCATGAAG

Plin2 GATTCATTCACGTGGCCTCT GGGAAGGAAAAACCTCACCT

Plin5 AGGGGACTAGACAAATTGG GCTTCTCCGACTTGCC

Enho CTCATCGCCATCGTCTGCAAT CGCACTGGATTCCGAGAGAGA

Tnf CATCTTCTCAAAAT TCGAGTGACAA TGGGAGTAGACAA GGTACAACCC

Il6 CCGGAGAGGAGACTTCACAGA AGAATTGCCATTGCACAACTCTT

Il1b CAACCAACAAGTG ATATTCTCCATG GATCCACACTCTC CAGCTGCA

Ccl2 AACTCTCACTGAAGCCAGCTCT CGTTAACTGCATCTGGCTGA

Cxcl9 TGAAGTCCGCTGTTCTTTTCC GGGTTCCTCGAACTCCACACT

Cxcl10 CCAGTGAGAATGAGGGCCATA CTCAACACGTGGGCAGGAT

Apoa4 CGTGCAGGAGAAACTCAACCA TCACCTTGCTCTGCACGTCTT

Lcn2 TTTCACCCGCTTTGCCAAGT GTCTCTGCGCATCCCAGTCA

Col1a1 TAGGCCATTGTGTATGCAGC ACATGTTCAGCTTTGTGGACC

Gfap TCCTGGAACAGCAAAACAAG CAGCCTCAGGTTGGTTTCA

Acta2 TCCTCCCTGGAGAAGAGCTAC TATAGGTGGTTTCGTGGATGC

Msln GCAGTCAGGGAGGTTCTGAGG GGTGGAGACTGACCACTTCGA

Ctgf GGGCCTCTTCTGCGATTTC ATCCAGGCAAGTGCATTGGTA

Ccn1/Cyr61 GGATCTGTGAAGTGCGTCCT CTGCATTTCTTGCCCTTTTT

Gli2 CAAGCAGAACAGCGAGTCAG TCAGCCTCAGTCTTGACC

Cers1 GCCACCACACACATCTTTCGG GGAGCAGGTAAGCGCAGTAG

Akr1b7 CCACCTTCGTGGAACTCAG CTTGGCCTGGGGAAGACT

Atf6 TGATCAATGGGCAGGACTATG GAACCAAAGAAGGTGCTGGTT

Ddit3 AAGCAACGCATGAAGGAGAAG TTCCGGAGAGACAGACAGGA

Hsp701a GGCCAGGGCTGGATTACT GCAACCACCATGCAAGATTA

Hsp701b GAAGACATATAGTCTAGCTGCCCAGT CCAAGACGTTTGTTTAAGACACTTT

Xbp1 CTTTTGGGCATTCTGGACAAG AGGTCCCCACTGACAGAGAAA

Xbp1p AGCCATTGTCTGAGACCACCT ACACTAATCAGCTGGGGGAAA

Cdkn2a GGGTTTTCTTGGTGAAGTTCG TTGCCCATCATCATCACCT

Ctnnb1 TGCAGATCTTGGACTGGACA AAGAACGGTAGCTGGGATCA

Ccnd1 TACTTCAAGTGCGTGCAGAAGG CAAGGGAATGGTCTCCTTCATC

Myc CCTAGTGCTGCATGAGGAGA TCCACAGACACCACATCAATTT

Mmp7 TAATTGGCTTCGCAAGGAGA AAGGCATGACCTAGAGTGTTCC

Ogg1 TTATCATGGCTTCCCAAACC GTACCCCAGGCCCAACTT

Ccl2 CATCCACGTGTTGGCTCA GATCATCTTGCTGGTGAATGAGT

Il1b TGTAATGAAAGACGGCACACC TCTTCTTTGGGTATTGCTTGG

Il12a CCAGGTGTCTTAGCCAGTCC GCAGTGCAGGAATAATGTTTCA

Il12b TTGCTGGTGTCTCCACTCAT GGGAGTCCAGTCCACCTCTAC

Tnf TCTTCTCATTCCTGCTTGTGG GGTCTGGGCCATAGAACTGA

Primer sets used for HFD-Asbt-/- experiments

Gene name Forward primer 5'-3' Reverse primer 5'-3'

Shp CGATCCTCTTCAACCCAGAT AGCCTCCTGTTGCAGGTGT

Mafg GACCCCCAATAAAGGAAACAA TCAACTCTCGCACCGACAT

Elovl6 CAGCAAAGCACCCGAACTA AGGAGCACAGTGATGTGGTG

Cgi58 ATCTTTGGAGCCCGATCCT CTTCTGGCTGATCTGCATACAC

Pgc1a GAAAGGGCCAAACAGAGAGA GTAAATCACACGGCGCTCTT

Acox1 CACCATTGCCATTCGATACA TGCGTCTGAAAATCCAAAATC

Ehhadh CCGGTCAATGCCATCAGT CTAACCGTATGGTCCAAACTAGC

Srebp2 ACCTAGACCTCGCCAAAGGT GCACGGATAAGCAGGTTTGT

Cidea AAACCATGACCGAAGTAGCC AGGCCAGTTGTGATGACTAAGAC

Scd1 TTCCCTCCTGCAAGCTCTAC CAGAGCGCTGGTCATGTAGT

Cyclophilin2 TTCTTCATAACCACAGTCAAGACC TCCACCTTCCGTACCACATC

Cyp7a1 AGCAACTAAACAACCTGCCAGTACTA GTCCGGATATTCAAGGATGCA

Srebp1 GGCTCTGGAACAGACACTGG TGGTTGTTGATGAGCTGGAG

Hmgr CTTGTGGAATGCCTTGTGATTG AGCCGAAGCAGCACATGAT

Acc1 TGGACAGACTGATCGCAGAGAAAG TGGAGAGCCCCACACACA

Fas GCTGCGGAAACTTCAGGAAAT AGAGACGTGTCACTCCTGGACTT

Ntcp GAAGTCCAAAAGGCCACACTATGT ACAGCCACAGAGAGGGAGAAAG

Abcb11 AAGCTACATCTGCCTTAGACACAGAAA CAATACAGGTCCGACCCTCTCT

Abcg8 AACCCTGCGGACTTCTACG CTGCAAGAGACTGTGCCTTCT

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