2. Academic Validation
  3. Hepatocyte free cholesterol lipotoxicity results from JNK1-mediated mitochondrial injury and is HMGB1 and TLR4-dependent

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.
Lay T Gan 1,  Derrick M Van Rooyen 1,  Mark E Koina 2,  Robert S McCuskey 3,  Narcissus C Teoh 1,  Geoffrey C Farrell 4
Affiliations

Affiliations

  • 1 Liver Research Group, Australian National University (ANU) Medical School at The Canberra Hospital, Garran, ACT, Australia.
  • 2 Department of Anatomical Pathology, ACT Pathology, The Canberra Hospital, ACT, Australia.
  • 3 Department of Cellular and Molecular Medicine, College of Medicine, University of Arizona, USA.
  • 4 Liver Research Group, Australian National University (ANU) Medical School at The Canberra Hospital, Garran, ACT, Australia. Electronic address: [email protected].
PMID: 25064435 DOI: 10.1016/j.jhep.2014.07.024
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.

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