title: Hepatocyte free cholesterol lipotoxicity results from JNK1-mediated mitochondrial injury and is HMGB1 and TLR4-dependent.
abstract: OBJECTIVE
  Free cholesterol (FC) accumulates in non-alcoholic steatohepatitis (NASH) but not in simple steatosis. We sought to establish how FC causes hepatocyte injury.
  
  
  METHODS
  In NASH-affected livers from diabetic mice, subcellular FC distribution (filipin fluorescence) was established by subcellular marker co-localization. We loaded murine hepatocytes with FC by incubation with low-density lipoprotein (LDL) and studied the effects of FC on JNK1 activation, mitochondrial injury and cell death and on the amplifying roles of the high-mobility-group-box 1 (HMGB1) protein and the Toll-like receptor 4 (TLR4).
  
  
  RESULTS
  In NASH, FC localized to hepatocyte plasma membrane, mitochondria and ER. This was reproduced in FC-loaded hepatocytes. At 40 μM LDL, hepatocyte FC increased to cause LDH leakage, apoptosis and necrosis associated with JNK1 activation (c-Jun phosphorylation), mitochondrial membrane pore transition, cytochrome c release, oxidative stress (GSSG:GSH ratio) and ATP depletion. Mitochondrial swelling and crystae disarray were evident by electron microscopy. Jnk1(-/-) and Tlr4(-/-) hepatocytes were refractory to FC lipotoxicity; JNK inhibitors (1-2 μM CC-401, CC-930) blocked apoptosis and necrosis. Cyclosporine A and caspase-3 inhibitors protected FC-loaded hepatocytes, confirming mitochondrial cell death pathways; in contrast, 4-phenylbutyric acid, which improves ER folding capacity did not protect FC-loaded hepatocytes. HMGB1 was released into the culture medium of FC-loaded wild type (WT) but not Jnk1(-/-) or Tlr4(-/-) hepatocytes, while anti-HMGB1 anti-serum prevented JNK activation and FC lipotoxicity in WT hepatocytes.
  
  
  CONCLUSIONS
  These novel findings show that mitochondrial FC deposition causes hepatocyte apoptosis and necrosis by activating JNK1; inhibition of which could be a novel therapeutic approach in NASH. Further, there is a tight link between JNK1-dependent HMGB1 secretion from lipotoxic hepatocytes and a paracrine cytolytic effect on neighbouring cholesterol-loaded hepatocytes operating via TLR4.
authors: Liver Research Group, Australian National University (ANU) Medical School at The Canberra Hospital, Garran, ACT, Australia.
authors: Lay T
authors: LT
authors: Gan
authors: Liver Research Group, Australian National University (ANU) Medical School at The Canberra Hospital, Garran, ACT, Australia.
authors: Derrick M
authors: DM
authors: Van Rooyen
authors: Department of Anatomical Pathology, ACT Pathology, The Canberra Hospital, ACT, Australia.
authors: Mark E
authors: ME
authors: Koina
authors: Department of Cellular and Molecular Medicine, College of Medicine, University of Arizona, USA.
authors: Robert S
authors: RS
authors: McCuskey
authors: Liver Research Group, Australian National University (ANU) Medical School at The Canberra Hospital, Garran, ACT, Australia.
authors: Narcissus C
authors: NC
authors: Teoh
authors: Liver Research Group, Australian National University (ANU) Medical School at The Canberra Hospital, Garran, ACT, Australia. Electronic address: geoff.farrell@anu.edu.au.
authors: Geoffrey C
authors: GC
authors: Farrell
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D024243:HMGB1 Protein / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D008930:Mitochondria, Liver / Q000378:metabolism*; D048055:Mitogen-Activated Protein Kinase 8 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D048056:Mitogen-Activated Protein Kinase 9 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D065626:Non-alcoholic Fatty Liver Disease / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D018384:Oxidative Stress / Q000502:physiology; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D024243:HMGB1 Protein; C582808:HMGB1 protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D002784:Cholesterol; D048056:Mitogen-Activated Protein Kinase 9; D048055:Mitogen-Activated Protein Kinase 8
keywords: Electron microscopy; Endoplasmic reticulum stress; Membrane pore transition; Mitochondrial oxidative stress; Plasma membrane