Volume 141, Issue 4, Pages 1393-1403.e5 (October 2011) Hepatic Free Cholesterol Accumulates in Obese, Diabetic Mice and Causes Nonalcoholic Steatohepatitis  Derrick M. Van Rooyen, Claire Z. Larter, W. Geoffrey Haigh, Matthew M. Yeh, George Ioannou, Rahul Kuver, Sum P. Lee, Narci C. Teoh, Geoffrey C. Farrell  Gastroenterology  Volume 141, Issue 4, Pages 1393-1403.e5 (October 2011) DOI: 10.1053/j.gastro.2011.06.040 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Increased hepatic cholesterol levels and LDLR expression in foz/foz mice with NASH. (A) Hepatic CE and (B) FC content in normal chow (0% [wt/wt] cholesterol) and HF (0.2% [wt/wt] cholesterol)-fed WT and foz/foz mice at 12 weeks (white bars) and 24 weeks (black bars) (n = 5–10 per group; see Materials and Methods) as determined by high-performance liquid chromatography. (C) LDLR protein expression, normalized to heat shock protein 90 (HSP90) expression. (D) Representative LDLR IHC staining from 24-week liver sections. Arrows indicate positive staining. Scale bars = 50 μm. *P < .05 vs diet-matched control. #P < .05 vs genotype-matched control. †P < .05 vs time-matched control. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Decreased cholesterol and BA biosynthesis as well as canalicular transporter gene expression in HF-fed foz/foz versus WT mice. (A) Microsomal HMGR activity at 12 weeks (white bars) and 24 weeks (black bars) in foz/foz and WT mice according to diet (values for n are in Materials and Methods). (B) ACAT-2 protein, (C) CE hydrolase (CEH) mRNA, and (D) Cyp7a1 mRNA expression at 12 and 24 weeks. (E) Hepatic bile salt exporter protein (Bsep) and (F) ATP-binding cassette protein G8 (ABCG8) protein expression at 12 and 24 weeks. Heat shock protein 90 (HSP90) (shown in F) was used as a loading control but not shown in all panels for clarity (results for loading controls were similar). Same mice as in Figure 1. *P < .05 versus diet-matched control. #P < .05 versus genotype-matched control. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Effect of diet and genotype on hepatic expression of nuclear regulators involved in cholesterol homeostasis. (A) SREBP-2, (B) LRH-1, (C) FXR, and (D) Shp expression at 12 weeks (white bars) and 24 weeks (black bars) was assessed using Western blotting of isolated hepatic nuclear protein. TATA-box binding protein (TBP) (shown in B) was used as a loading control. Same mice as preceding figures. *P < .05 versus diet-matched control. #P < .05 versus genotype-matched control. †P < .05 versus time-matched control. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Insulin alters cholesterol-regulating protein expression in primary hepatocyte cultures. (A) Levels of SREBP-2, (B) LDLR, and (C) bile salt exporter protein (Bsep) expression in primary hepatocytes (whole cell lysates) treated with 0, 0.2, 6.5, and 13.0 ng/mL insulin for 48 hours (n = 3/group). As shown in D, protein expression was normalized to β-actin. (E) LRH-1 and (F) Shp mRNA were assessed by reverse-transcription polymerase chain reaction. There was insufficient material to prepare nuclear protein extracts. This experiment was conducted 3 times with analyses in triplicate (n = 9 per group total). *P < .05 between groups. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Dietary cholesterol modulates hepatic cholesterol content and liver injury but not other lipid profiles in NASH. (A) Serum ALT, (B) total hepatic CE, and (C) hepatic FC content in WT (white bars) and foz/foz (black bars) mice (n values as per Materials and Methods) fed HF diet containing 0, 0.2%, or 2.0% (wt/wt) cholesterol for 24 weeks. (D) Hepatic triglycerides (TG), (E) diacylglycerides (DAG), and (F) total FFAs as determined by high-performance liquid chromatography. *P < .05 versus diet-matched control. #P < .05 versus genotype-matched, 0.0% cholesterol group. †P < .05 versus genotype-matched, 0.2% cholesterol group. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Dietary cholesterol modulates hepatocyte apoptosis and macrophage recruitment in NASH. (A) Cell death, as assessed by cytokeratin-18 (Ck-18) fragmentation, and (B) macrophage cell recruitment (F4/80) were determined using IHC detection to (C) quantify positive cells (Materials and Methods). (D) Quantification (ImageJ) of representative Sirius red–stained liver sections (E) from WT (white bars) and foz/foz (black bars) mice (n values as per Materials and Methods) fed HF diet containing 0, 0.2%, or 2.0% (wt/wt) cholesterol for 24 weeks. Arrows indicate positive staining. Same livers as in Figure 5. Scale bars = 20 μm (C) and 500 μm (E). *P < .05 versus diet-matched control. #P < .05 versus genotype-matched, 0.2% cholesterol group. †P < .05 versus genotype-matched, 2.0% cholesterol group. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Differentially regulated pathways involved in cholesterol homeostasis. In addition to the pathways illustrated in Figure 2, we studied (A) SR-B1 protein expression and (B) Cyp27a1, (C) Cyp7b1, and (D) Cyp8b1 mRNA expression at 12 weeks (white bars) and 24 weeks (black bars) in WT and foz/foz mice (n values as per Materials and Methods). (E) Bile salt exporter protein (Bsep) IHC staining in 24-week WT and foz/foz mouse livers produced similar results to Western blot analyses (Figure 2). (F) ABCG5 protein expression in mice at 12 and 24 weeks. Scale bars = 50 μm. Arrows indicate positive staining. *P < .05 versus diet-matched control. #P < .05 versus genotype-matched, 0.2% cholesterol group. †P < .05 versus genotype-matched, 2.0% cholesterol group. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Dietary cholesterol increases serum cholesterol levels but fails to alter other lipid profiles in HF-fed WT and foz/foz mice. (A) Total serum cholesterol and (B) triglyceride (TG) levels in WT (white bars) and foz/foz (black bars) mice (n values as per Materials and Methods) fed HF diet containing 0.0%, 0.2%, or 2.0% (wt/wt) cholesterol for 24 weeks. (C) Monoacylglycerides (MAG), (D) saturated FFAs (SaFA), (E) monounsaturated FFAs (MuFA), and (F) polyunsaturated FFAs (PuFA) were determined by high-performance liquid chromatography. *P < .05 versus diet-matched control. #P < .05 versus genotype-matched, 0.0% cholesterol group. †P < .05 versus genotype-matched, 0.2% cholesterol group. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Comparison between LDLR localization in foz/foz, methionine, and choline-deficient (MCD) and carbon tetrachloride–treated mice. LDLR localization in (A) chow-fed and (B) HF-fed foz/foz mice at 24 weeks, (C) MCD control and (D) MCD-deficient mice at 8 weeks, and (E) carbon tetrachloride control and (F) treated mice at 4 weeks. Scale bars = 20 μm. Gastroenterology 2011 141, 1393-1403.e5DOI: (10.1053/j.gastro.2011.06.040) Copyright © 2011 AGA Institute Terms and Conditions