Results: In livers from obese, diabetic mice with NASH, FC co-localised to plasma membrane, mitochondria and, to lesser extent, endoplasmic reticulum (ER). This pattern was replicated www.selleckchem.com/products/VX-809.html in primary hepatocytes incubated with LDL, which dose-dependently increased hepatocyte FC. Such FC loading caused dose-dependent increases in LDH
leakage, apoptosis (Höechst 33342) and necrosis (propidium iodide; release of high mobility group box1 [HMGB1]). At 40 μM LDL, cell death was associated with JNK1 activation (c-Jun phosphorylation), mitochondrial outer membrane pore transition (reduced tetramethyl rhodamine methyl ester fluorescence) resulting in cyt c release into cytoplasm, cellular oxidative stress (increased GSSG:GSH ratio) and ATP depletion. JNK inhibition by 1–2 μM CC-401 or CC-930 ameliorate FC-induced apoptosis and necrosis. Similarly, JNK1–/– primary hepatocytes
were refractory to FC-induced injury. Cyclosporine A (10 μM) and caspase-3 check details inhibition also protected WT hepatocytes from FC-mediated injury/cell death, but 500 μM 4-phenylbutyric acid (ER chaperone) had no effect and there was no evidence of ER stress in in vitro or in intact livers. FC deposition reduced plasma membrane fluidity (by pyrene eximer-to-monomer fluorescence emission), while blebbing and fragmentation/release of MPs from the surface of FC-injured hepatocytes was evident on TEM and SEM. Finally, addition of HMGB1-enriched culture medium or MP fractions from FC-loaded hepatocytes activated resting
KCs, as assessed by nuclear translocation of NF-κB, release of IL-1β, TNF-α and ultra-structural changes. Conclusions: These highly novel findings demonstrate how FC deposition in mitochondria and plasma membrane causes apoptosis and necrosis, confirm the centrality of JNK-1 activation for hepatocyte lipotoxic injury, and reveal direct links (via HMGB1 and MPs) between cholesterol lipotoxicity and engagement of KC activation/inflammatory recruitment in the transition of steatosis to NASH. 1. Van Rooyen DM, Larter CZ, Farrell GC et al. Hepatic Free Cholesterol Accumulated in Obese, Diabetic Mice and Causes Nonalcoholic Steatohepatitis. Gastroenterology 2011, 141(4), 1393–1403. 2. DM van Rooyen, Gan LT, Farrell Org 27569 GC et al. Pharmacological cholesterol lowering reverses fibrotic NASH in obese diabetic mice. J Hepatol 2013;Mar 7. doi:pii: S0168-8278(13)00146-3. 10.1016/j.jhep.2013.02.024. KR MULLER,1 NS EYRE,1 KH VAN DER HOEK,1 K LI,2 MR BEARD1 1Hepatitis C Research Laboratory, University of Adelaide, South Australia, 2Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN, USA Introduction: Only a small proportion of cells in the hepatitis C virus (HCV)-infected liver harbour replicating HCV.