Hedgehog Controls Hepatic Stellate Cell Fate by Regulating Metabolism Yuping Chen, Steve S. Choi, Gregory A. Michelotti, Isaac S. Chan, Marzena Swiderska-Syn, Gamze F. Karaca, Guanhua Xie, Cynthia A. Moylan, Francesca Garibaldi, Richard Premont, Hagir B. Suliman, Claude A. Piantadosi, Anna Mae Diehl  Gastroenterology  Volume 143, Issue 5, Pages 1319-1329.e11 (November 2012) DOI: 10.1053/j.gastro.2012.07.115 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Metabolism is reprogrammed during HSC transdifferentiation. Primary HSCs were cultured for 7 days. Culture-related changes in (A) mRNA from the glycolytic enzymes hexokinase (HK2), phosphofructokinase (PFKP), and pyruvate kinase M2 (PKM2), and (B and C) protein expression of PKM2, HK2, and monocarboxylate transporter 4 (MCT4). Related changes in (B) the MF marker (α-SMA), (D) glucose transporter (GLUT1) and lactate transporter MCT4, (E) the gluconeogenesis-related genes phosphoenolpyruvate carboxykinase (PCK1), and fructose-1,6-bisphosphatase-1 (FBP1), and (F) intracellular lactate. **P < .01; ***P < .001 vs day 0. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Metabolic reprogramming controls the fate of HSCs. Seven-day cultured primary HSCs were analyzed after 3 days of treatment with (A and B) 2DG (2.5 mmol/L) to inhibit glycolysis or (C and D) FX11 (20 μmol/L, LDHA inhibitor) to block lactate generation. Immunocytochemistry was used to compare effects of (A) 2DG or (C) FX11 on proliferation (BrdU incorporation), expression of MF markers (α-SMA), and lipid content (Oil Red O). (B and D) Changes in gene expression were assessed by qRT-PCR. ***P < .001 vs day 0; †††P < .001 vs day 7 control. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Hh signaling controls metabolic reprograming to direct the fate of HSCs. (A) qRT-PCR analysis of freshly isolated HSCs from SMO-LoxP mice that were infected with adenoviral GFP or Cre 2 days before HSC isolation SMO KD and freshly isolated rat primary HSCs treated with SAG (Hh agonist, 0.3 μmol/L) for 24 hours (results are expressed as % change of adenoviral GFP infected control). (B) qRT-PCR analysis and immunostaining of murine HSCs treated with GDC-0449 on culture days 4 through 7 (to inhibit SMO) or culture day 7 SMO-LoxP HSCs treated with adenoviral Cre on day 4 (to disrupt the Smo gene). (C) qRT-PCR analysis of HIF1α mRNA in primary rat HSCs and (D) 8B cells transfected with equal amounts of expression constructs for GLI1 or GLI2 (or empty vector) 2 days earlier. (E) Chromatin was immunoprecipitated from LX2 cells using GLI2 or immunoglobulin G antibodies and analyzed by PCR. The GLI consensus sequence within the chromatin immunoprecipitation amplicon is in bold and underlined. GFAP was used as a specificity control. (F) qRT-PCR analysis of culture day 7, HSCs treated with acriflavine (ACF; HIF1α inhibitor) for 3 days. *P < .05; **P < .01; ***P < .001 vs control. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Metabolic reprogramming of HSCs is a conserved response to liver injury. Mice were (A and B) injected once with CCl4, (C and D) fed methionine-choline–deficient (MCD) diets for 8 weeks, or (E and F) subjected to BDL for 2 weeks to cause liver damage. Changes in glycolysis-related gene/protein expression were evaluated by (A, C, and E) qRT-PCR, (B, D, and F) immunoblot, and (B, D, and F and Supplementary Figure 3) immunohistochemistry and correlated to changes in MF marker expression. *P < .05; **P < .01; ***P < .001 vs control. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Hh signaling controls metabolic reprogramming during liver injury in vivo. Immunohistochemistry identifies cells expressing the M2 isozyme of PKM2, a specific marker of glycolytic activity, in (A) healthy adult mice and (B–D) different liver injury models. Hh signaling was (B) inhibited in PH mice by cyclopamine, (C) inhibited in aged MDR2−/− mice by GDC-0449, or (D) abrogated selectively in MFs by treating DTG mice with tamoxifen, as indicated. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Hh controls the fate of HSCs by regulating metabolism. Hh ligands released from dying hepatocytes activate Hh signaling in Q-HSCs. This inhibits lipogenesis and gluconeogenesis while activating aerobic glycolysis (the Warburg effect). The resultant glycolytic end products reprogram HSCs into proliferative MFs. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Metabolism is reprogrammed during HSC transdifferentiation. Primary Q-HSCs were cultured for 7 days, during which time they transitioned into MFs. qRT-PCR was performed to examine culture-related changes in mRNAs encoding (A) the glycolysis-related enzymes, phosphoglycerate mutase (PGAM)1, phosphoglycerate kinase (PGK)1, enolase (ENO)1 and ENO2, (B) pyruvate dehydrogenase kinase (PDK)3, an enzyme that inhibits conversion of pyruvate to acetyl coenzyme A, (C) representative genes involved in lipogenesis (PPARγ), lipid oxidation (PPARα), and lipid uptake (FABP4), (D) typical markers of MF, and (E) several cell cycle–related genes. Data are presented as mean ± SEM. *P < .05, ***P < .001 vs Q-HSCs (culture day 0). Immunostaining shows differences in expression of MF markers (α-SMA), proliferative activity (BrdU incorporation), and lipid content (Oil Red O) of freshly isolated Q-HSCs and culture day 7 MF-HSCs (F). Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Glycolytic enzyme gene expression is rapidly induced during the initial 24 hours of HSC culture. Primary rat HSCs were cultured for 24 hours and harvested at different time points for qRT-PCR assay. *P < .05, ***P < .001 vs freshly isolated Q-HSCs (0 hour). Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Metabolism is reprogrammed in HSCs cultured in low glucose medium. Primary rat HSCs were cultured in Dulbecco's modified Eagle medium containing low glucose (1000 mg/L) for 7 days. Changes in (A) mRNA and (B and C) protein expression of genes encoding glycolytic enzymes, transporters, gluconeogenic FBP1, and MF marker (α-SMA) were evaluated. *P < .05, **P < .01, ***P < .001 vs Q-HSCs (culture day 0). Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Increase in mitochondria during HSC transdifferentiation. (A) Representative electron micrographs of day 0 rat HSCs. Arrows indicate mitochondria. Scale bar = 4 μm. Mitochondria number per cell was quantified by counting mitochondria in freshly isolated day 0 HSCs and day 7 culture-activated HSCs (at least 15 cells per group). Data are expressed as mean ± SEM, P < .001 vs day 0. (B) Primary HSCs were isolated from mtGFP-tg mice, cultured, and observed under a 40× fluorescence microscope daily. Representative images are shown. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Inhibiting lactate generation from pyruvate blocks metabolic reprogramming in HSCs. (A) Treating MF-HSC 8B cells with an LDHA inhibitor (FX11, 10 or 20 μmol/L) or vehicle control (dimethyl sulfoxide) for 2–3 days to block the conversion of pyruvate to lactate has little effect on cell growth but significantly inhibits expression of MF markers as assessed by (B) qRT-PCR and (C) immunoblot. Data are presented as mean ± SEM. **P < .01, ***P < .001 vs control treatment. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 Fibrogenesis is a conserved response to liver injury. Mice were (A) given a single injection of CCl4 to induce acute liver injury, (B) fed methionine-choline–deficient (MCD) diets for 8 weeks, or (C) subjected to BDL for 2 weeks to cause chronic liver damage. (A–C) Repair-related changes in expression of myofibroblast markers (α-SMA, vimentin, and S100A4) and collagen 1α1 were evaluated by qRT-PCR. Data are presented as mean ± SEM. *P < .05, **P < .01, and ***P < .001 vs control treatment. (D) Liver extracts of mice subjected to sham vs BDL were separated with polyacrylamide gel electrophoresis, and Ponceau solution was used to stain the gel to show equal protein loading. BDL-induced changes in MF markers and glycolytic enzymes were evaluated by immunoblot (Figure 4). Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 7 HSCs become glycolytic in injured livers. Double staining for PKM2 (brown) and desmin (green) in various animal models of liver injury shows that cells expressing the HSC marker desmin coexpress PKM2, a marker of glycolytic cells. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 8 Glycolytic stromal cells accumulate in human liver diseases. (A) Compared with normal healthy livers (NHL), livers from patients with primary biliary cirrhosis (PBC), nonalcoholic steatohepatitis (NASH), and alcoholic steatohepatitis (ASH) showed increased expression of genes encoding the glycolytic enzymes HK2 and PFKP. (B) Accumulation of stromal cells expressing the glycolysis marker PKM2 was also increased in patients with these and other types of liver disease, including biliary atresia (BA), progressive familial intrahepatic cholestasis (PFIC), and chronic hepatitis C (HCV). (C) qRT-PCR analysis of PKM2 expression in liver RNA from patients with nonalcoholic steatohepatitis who had mild fibrosis (Brunt stage F0–1) or severe fibrosis (Brunt stage F3–4; n = 16/group). Data are presented as mean ± SEM. *P < .05, **P < .01 vs NHL. Gastroenterology 2012 143, 1319-1329.e11DOI: (10.1053/j.gastro.2012.07.115) Copyright © 2012 AGA Institute Terms and Conditions