Cell Metabolism, Volume 27

Supplemental Information

Digoxin Suppresses Pyruvate Kinase M2-Promoted

HIF-1a Transactivation in Steatohepatitis

Xinshou Ouyang, Sheng-Na Han, Ji-Yuan Zhang, Balazs Tamas Nemeth, PalPacher, Dechun Feng, Ramon Bataller, Joaquin Cabezas, Peter Stärkel, JoanCaballeria, Rebecca LePine Pongratz, Shi-Ying Cai, Bernd Schnabl, Rafaz Hoque, YonglinChen, Wei-hong Yang, Irma Garcia Martinez, Fu-Sheng Wang, Bin Gao, Natalie JuliaTorok, Richard Glenn Kibbey, and Wajahat Zafar Mehal

Figure S1. Related to Figure 1. The myeloid-specific lysozyme-M cre-recombinase driven HIF1α null mice (HIF-1a-cKO) show significant reduction of liver injury (A), PKM2 is highly expressed in mouse myeloid cells (B). (A) The reduction of serum level of alanine transaminase (ALT) in HIF-1a-cKO mice that were treated with LPS/D-GalN for 6 h. Data represent mean ± SD (n=4-5). *p <0.05, **p<0.01. (B) PKM2 gene expression shows high level in myeloid cells compared with hepatocytes. The figure was citied form Gene Expression Atlas Database (https://www.ebi.ac.uk, Array Expression: experiment E-MTAB-3578) with slight modification. TPM (Transcripts Per Million).

Figure S2. Related to Figure 2 and 3. Digoxin limits alcoholic hepatic steatosis and inflammation in mouse model (A-D), and the expression of HIF-1a signature genes in the livers of patients with early and severe alcoholic hepatitis (E-I). C57BL/6 mice were placed on a high alcohol diet concurrently with intraperitoneal injection of digoxin (0.2, 0.5 or 1 mg/kg) or vehicle control daily for 10 days. Liver tissues were examined by H & E staining (A). Neutrophils MPO positive staining was assayed, and quantification of positive MPO cells per field was determined (B). Serum ALT, AST levels in digoxin versus vehicle control were measured (C, D). All data throughout the figure are shown as the mean ± SD from 11-5 mice in each group. *p <0.05.

The expression levels of HIF-1a (E) and HIF-1a dependent (F-I) genes were compared between the livers of patients with early and severe alcoholic hepatitis. The clinical characterization of early NASH (Early) and Severe Alcoholic Hepatitis (Severe) patients is described in Table S1. Expression of selected genes was obtained from normalized unpublished RNA-seq data from liver samples from the Human Biorepository Core of the NIAAA-funded InTeam Consortium (www.inteamconsortium.org). Libraries were sequenced on an Illumina HiSeq2000 and sequences mapped to the GRCh37/hg19 human reference. Values are given as actual numbers or means ± SD. Data were compared by Student’s T test, Mann-Whitney test or Chi-square test.

Figure S3. Related to Figure 2 and 3. Effect of chronic digoxin treatment on heart rate, cardiac electrophysiology and cardiac performance (A) Representative ECG recordings demonstrate the absence of effect of 1 mg/kg i.p. (2 x / week) digoxin on heart rate and ECG complex morphology at the end of the 3-week treatment period. Similar results were observed at each of the 12 time points investigated during the 3 weeks’ treatment period. Below, typical arrhythmia and ECG morphology is shown 30 mins following administration of a high/toxic dose of Digoxin 10 mg/kg. These mice were euthanized, because of the severe arrhythmias.

(B) Representative echocardiography short axis M-mode images taken at the end of the treatment period. Note the similar heart rate and chamber diameters among groups studied. LVIDd – left ventricular internal dimension in diastole; LVIDs – left ventricular internal dimension in systole.

(C) The effect of chronic Digoxin treatment 1 mg/kg i.p. for 3 weeks (2 x / week) or vehicle is shown on heart rate, ECG morphology (PR interval and QRS width).

(D) The effect of chronic Digoxin treatment 1 mg/kg i.p. for 3 weeks (2 x / week) or vehicle is shown on cardiac performance (ejection fraction and fractional shortening) measured by echocardiography. ECG and echocardiography measurements were conducted 4x/week: 8 and 48 hours following each Digoxin administration for a period of 3 weeks. All values are expressed as mean ± S.E.M. of 8 experiments/group. Note, chronic treatment with Digoxin 2 x 1 mg/kg/week i.p. had no cumulative toxicity or any significant effects compared to vehicle on variables studied.

B

A

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Figure S4. Related to Figure 6. Identify digoxin bound proteins. (A) Synthetic scheme of digoxin immobilized affinity resin. In a BioRad PolyPrep reaction vessel, a total volume of 5 mL was reacted diisocyanato hexane and Tentagel-NH2 resin to provide isocyanate functionalized beads. Ninhydrin staining confirmed the coupling of the diisocanatohexane. The isocyanate functionalized beads were then reacted with digoxin. Between each synthetic step, the beads were washed 3x 5 mL DMF. The isocyanate functionalized beads were then reacted with the –OH groups of digoxin. After digoxin coupling the beads were washed 3x with DMF, 3 x MeOH, 3x DCM and stored at 4°C in DCM. Digoxin negative beads were prepared by quenching the isocyanate functionalized beads with 2 eq methylamine (Sigma) 0.1% DMAP in DMF O/N and washed 3 x with DMF, 3 x MeOH, 3x DCM and stored at 4°C in DCM (B). The raw264.7 cells were treated with digoxin for 3 h, and then LPS for 16 h. The total cell lysates were incubated with control- or immobilized affinity resin for overnight. The bead un-bound proteins were removed by extensive washing. The bead bound protein complexes were then eluted by acidic buffer. The total elute protein complexes were applied on SDS-PAGE, and then silver staining.

LPS stimulated macrophages Preparation of Digoxin functionalized beads

KDa+-+-

Beads

70

CT Digoxin

LPS

A B

A

B

Figure S5. Related to Figure 6 and 7. Digoxin does not influence PKM2 nuclear translocation (A), and TEPP-46 reduces endotoxin-induced hepatic damage (B). (A) Raw264.7 cells were pre-treated with digoxin for 3 h, and then LPS for 6 h. The cells were fixed, and performed immunofluorescence staining with anti-PKM2 antibody, and the nucleus was stained with DAPI. (B) Mice were injected intraperitoneally with TEPP-46 (50 mg/kg) or vehicle for 1h, followed by LPS-D-GalN for further 6 h. (B-left) Reduced hemorrhage and cellular necrosis in liver tissues by TEPP-46 from H&E staining, (B-right) Reduced serum ALT level by TEPP-46. Data represent mean ± SD from 5-6 mice. Scale bar, 100µm.

Figure S6. Related to Figure 6E-P. The CG analogs reduce HRE promoter activity and digoxin does not directly influence LXR, FXR and PPARa promoter activity. HEK293 T cells were co-transfected with PKM2 and HRE promoter luciferase constructs and treated with the CGs of Proscillaridin A and Digitonin, and the controls of Cholic acid and Obeticholic acid (A). HEK293 T cells were co-transfected with LXR- (B), FXR- (C), PPARa (D), luciferase constructs in the presence of common RXR plasmid and individual consensus response element plasmid, and then stimulated cells with the agonists of 22(R)-OH- cholesterol (OH) (B), Chenodeoxycholic acid (CDCA) (C) and Wy-14643 (WY) (D) in the presence or absence of digoxin. The cells were harvested in 18 h after stimulation. The luciferase activity was measured by dual-luciferase reporter assay and normalized to renilla control.

0

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luc

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lold

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LXR - - - + + + + + +OH - - - - + + +- -

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050100150200250

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WY - - - - + + +- -Digoxin - - +

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FXR - - - + + + + + +CDCA - - - - + + +- -Digoxin - - +

B C D

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PKM

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Proscillaridin A

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A

Figure S7. Related to Figure 7. Gen-network analysis for the association of PKM2 with HIF-1a (A), and PKM2 with histones (B) based on STRING program (A). PKM2 has a related signaling with HIF-1a including EP300, EPAS1, VHL and UBC based on an analysis of the STRING database (A). PKM2 has a related signaling with histones including his2A and his3A based on an analysis of the STRING database (B).

Table S1. Related to Figure 2 and 3. Phenotypes of Early vs Severe Alcoholic Hepatitis

EarlyASH[n=12]

SevereAlcoholicHepatitis[n=18]

p-value

Gender(male/female) 8/4 11/7 0.757Age(years-old) 53.1±7.8 50.4±10.3 0.447AST(IU/dL) 109±49 158±59 0.026ALT(IU/dL) 70±19 52±30 0.084GGT(IU/dL) 477±338 337±220 0.081AP(IU/dL) 100±44 441±196 <0.001Bilirubin(mg/dL) 1.0±0.4 19.4±8.4 <0.001Albumin(g/dL) 4.4±0.3 2.6±0.4 <0.001INR 0.98±0.09 1.79±0.45 <0.001Creatinine(mg/dL) 0.69±0.19 1.36±1.4 0.014*Glucose(mg/dL) 107.5±33.3 102.8±38.2 0.733Cholesterol(mg/dL) 222±41 127±43 <0.001Triglycerides(mg/dL) 205±264 156±57 0.560Leucocytecount(x103/uL) 6.0±2.0 10.5±3.7 0.001Neutrophilcount(x103/uL) 4.2±2.0 8.7±3.5 <0.001Hemoglobin(g/dL) 14.8±1.5 11.0±2.9 <0.001Plateletscount(x103/uL) 179.5±61.9 130.7±69.4 0.060FibrosisStage:

- No fibrosis/Portal fibrosis - Expansive periportal

fibrosis - Bridging fibrosis/Cirrhosis

435

----18

0.001

MELD 7±1 25±5 <0.001AST,aspartateaminotransferase;ALT,alanineaminotransferase;GGT,gamma-glutamyltranspeptidase;AP,alkalinephosphatase;INR,internationalnormalizedratio;MELD,modelofend-stageliverdisease.

Valuesaregivenasactualnumbersormeans±SD.DatawerecomparedbyStudent’sTtest,Mann-Whitney*testorChi-squaretest.

EarlyASH:Patientswithalcoholicliverdisease(biopsy-provenalcoholicsteatohepatitis)withnopreviousepisodeofdecompensation/jaundice.Patientsconsumingmorethan60gramsperdayofethanol.

SevereAlcoholicHepatitis:Patientswithbiopsy-provenalcoholichepatitisfeaturespresentingwithanAge,serumBilirubin,INR,andserumCreatinine(ABIC)scoreabove6.71.

Patientswithfullycompensatedalcoholicsteatohepatitis(earlyASH,n=12)andpatientswithclinicalandanalyticalcriteria of severe alcoholic hepatitis (severe alcoholic hepatitis, n= 18) were included as described previously(JanssensFetal.JClinGastroenterol2010andAffoSetal,Gut2013).Theprotocolwasconformedtotheethicalguidelinesofthe1975DeclarationofHelsinkiandpatientssignedinformedconsentwereincluded.