MITOCHONDRIAL OXIDANT STRESS IN EXPERIMENTAL NON‐ALCOHOLIC STEATOHEPATITIS (NASH)

Abstract

Background and Hypothesis: The hypothetical two hits leading to NASH are insulin resistance and oxidative stress related to steatosis. Experimental NASH can be produced by feeding mice a methionine and choline deficient (MCD) diet, and exposing mouse AML-12 hepatocytes in vitro to MCD medium stimulates PI3-K dependent steatosis and release of ALT, both without global oxidative stress. Early mitochondrial swelling suggests that focal oxidative stress may be involved. We hypothesize that mitochondrial reactive oxygen species (mROS) generation leads to impaired beta-oxidation, accumulation of toxic acyl-CoA moieties and subsequent mitochondrial dysfunction. Further toxic acyl-CoA moieties may be a therapeutic target for supplementation with carnitine. Methods: AML-12 hepatocytes were exposed to MCD and control (MC+) media ± 0.5 mM L-carnitine (CARN), and mROS generation was evaluated by co-staining with fluorescent dyes, dihydroethidium (DHE, a ROS specific vital dye) and DAPI for double-stranded DNA. ROS generation was confirmed by a cell permeable superoxide scavenger, Manganese benzoyl porphyrin chloride (MnTBAP). Female A/J mice were fed MCD or MC+ diet ± 0.5% CARN in drinking water. mROS was measured by DHE staining in frozen liver sections obtained on days 3, 7 and 14. Results: DHE staining showed marked ↑ mROS activity within 30 min of exposure of AML-12 hepatocytes to MCD medium, which dissipated by 3 hr MCD exposure to levels in MC+ medium treated cells. Incubating with MnTBAP before DHE staining reduced fluorescence, confirming mROS generation. Supplementing with CARN substantially reduced mROS generation in MCD treated cells as evidenced by ↓ DHE fluorescence. ↑ hepatic mROS in MCD diet fed mice at all time points was reduced by CARN supplementation as evidenced by ↓ DHE fluorescence. Conclusions: 1. Focal mitochondrial oxidative stress due to ↑ mROS generation has a potentially important mechanistic role in experimental NASH. 2. CARN may provide a targeted strategy to protect from mROS stress and thus retard progression of NASH.