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1 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* The PDF file includes: *Corresponding author. Published 21 September 2016, Sci. Transl. Med. 8, 357ra122 (2016) DOI: /scitranslmed.aaf4823 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.

2 Legend for table S7 References (42 53) Other Supplementary Material for this manuscript includes the following: (available at 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).

3 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 ), 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%

4 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,

5 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 paraffinembedded 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

6 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 (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).

7 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% CO 2 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 (10 5 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.

8 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 posthoc 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.

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10 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.

11 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.

12 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.

13 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.

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15 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.

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19 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.

20 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.

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22 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.

23 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 αtmca ßTMCA TCA TCDCA TDCA Table S4: Differentially expressed genes and pathway analysis (provided as an Excel file).

24 Table S5: HFD composition and macronutrient information. Component g/kg Casein DL-Methionine 3.4 Sucrose Corn starch 80.0 Maltodextrin Hydrogenated vegetable oil (Primex) Soybean oil 10.0 Cholesterol 2.0 Cellulose 50.0 Mineral mix, AIN-93G-MX 46.0 Calcium Phosphate, dibasic 3.3 Niacin Calcium pantothenate Pyridoxine HCl Thiamin HCl Riboflavin Folic Acid Biotin Vitamin B12 (0.1% in mannitol) Vitamin E, DL-alpha tocopheryl acetate (500 IU/g) Vitamin A Palmitate (500,000 IU/g) Vitamin D3, cholecalciferol (500,000 IU/g) Vitamin K1, phylloquinone Choline bitartrate TBHQ, antioxidant % kcal from Chow HFD Protein Carbohydrate Fat

25 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

26 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

27 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

28 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).