Hepatocyte free cholesterol lipotoxicity results from JNK1-mediated mitochondrial injury and is HMGB1 and TLR4-dependent

J Hepatol. 2014 Dec;61(6):1376-84. doi: 10.1016/j.jhep.2014.07.024. Epub 2014 Jul 24.

Abstract

Background & aims: 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.

Keywords: Electron microscopy; Endoplasmic reticulum stress; Membrane pore transition; Mitochondrial oxidative stress; Plasma membrane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Apoptosis / physiology
  • Cells, Cultured
  • Cholesterol / metabolism*
  • Cytochromes c / metabolism
  • Disease Models, Animal
  • Female
  • HMGB1 Protein / metabolism*
  • Hepatocytes / metabolism*
  • Hepatocytes / pathology
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mice, Mutant Strains
  • Mitochondria, Liver / metabolism*
  • Mitogen-Activated Protein Kinase 8 / deficiency
  • Mitogen-Activated Protein Kinase 8 / genetics
  • Mitogen-Activated Protein Kinase 8 / metabolism*
  • Mitogen-Activated Protein Kinase 9 / deficiency
  • Mitogen-Activated Protein Kinase 9 / genetics
  • Mitogen-Activated Protein Kinase 9 / metabolism
  • Non-alcoholic Fatty Liver Disease / metabolism
  • Non-alcoholic Fatty Liver Disease / pathology
  • Non-alcoholic Fatty Liver Disease / physiopathology
  • Oxidative Stress / physiology
  • Toll-Like Receptor 4 / deficiency
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*

Substances

  • HMGB1 Protein
  • HMGB1 protein, mouse
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4
  • Adenosine Triphosphate
  • Cytochromes c
  • Cholesterol
  • Mitogen-Activated Protein Kinase 9
  • Mitogen-Activated Protein Kinase 8