Winning Abstracts from the 2006 National Medical Student Poster Competition: Mitochondrial Injury and Non-alcoholic Steatohepatitis.
Debasish Sundi, Northwestern University, Feinberg School of Medicine, 2008
Non-alcoholic steatohepatitis (NASH) is a disease of the liver linked to obesity and diabetes. The main cellular features of NASH are steatosis, inflammation, and fibrosis. 30% of 1.6 million children that have fatty liver disease in the U.S. are thought to have NASH. The mechanism by which NASH evolves and progresses is unknown; this is a barrier to revealing pharmacologic targets that can prevent or treat NASH. The goal of my research is to confirm the presence reactive oxygen species (ROS) as an important cell signal in an in vivo model of NASH. An in vivo murine model of NASH is the Methionine- and Choline-Deficient (MCD) diet. The extent of oxidation within mitochondria is measured by aconitase activity, which decreases upon oxidation of its 4Fe-4S active site cluster. NADPH fluorescence at 340 nm is a proxy for aconitase activity. We hypothesize that in our model of NASH, increases in ROS cause aconitase activity to decrease.
6-8 weeks old female A/J mice were divided into control and experimental groups. Control mice received a standard laboratory diet. Experimental mice received the MCD diet. Livers were excised and snap frozen in liquid nitrogen and stored at -80 C° until assay. 100 mg liver tissue per sample was homogenized with 50 μl protease inhibitor and 950 μl homogenization buffer (65 mM Tris HCl pH 7.5, 1.0 mM Sodium Citrate, 0.5 mM MgCl2). Each homogenate was centrifuged at 800g for 10 minutes at 4 C° to pellet out the crude nuclear fraction. The supernatant was centrifuged at 15000g for 15 minutes at 4 C° to form a mitochondrial pellet, which was subsequently re-suspended with 500 μl homogenization buffer and sonicated on ice for 20 seconds, twice. Protein concentration was estimated using a modification of the standard Bradford method that uses a protein precipitation step that eliminates interference from lipids (Pande and Murthy 1994, Anal Biochem). Precipitation reagents were 80% ethanol, 250 mM CaCl2, and 500 mM K3PO4. 5 μg of mitochondrial protein was added in a test tube to 3.4 ml PBS, 200 μl 10 mM Sodium Citrate, 200 μl 1.0 mM NADP, and 200 μl 1.0 U/ml Isocitrate Dehydrogenase. NADPH production was read at 340 nm on a Sequoia-Turner 112 fluorometer.
For fluorescence microscopy, AML-12 hepatocytes grown to 70% confluence were made quiescent with serum free medium for 24 hours. Cells were exposed to MCD or control media for 10 min to 3 hours. ROS was evaluated by co-staining with fluorescent dyes, dihydroethidium (DHE, a ROS specific vital dye) and DAPI for double-stranded DNA. A cell permeable superoxide scavenger, Mn (III) tetrakis (4-benzoic acid) porphyrin chloride was used as a negative control. Pre-incubation for 22 hours with 3-Nitropropionic acid, a known mitochondrial toxin, was used as a positive ROS control.
An assay for aconitase activity was successfully developed and implemented. There are not distinct differences in aconitase activity among control and MCD samples at time points ranging from 2 to 21 days. However, DHE staining showed marked ROS activity in the cytoplasm of MCD-treated AML-12 hepatocytes peaking at 30 minutes, compared to control cultures.
The in vivo aconitase assay did not demonstrate increased ROS in livers of mice fed a NASH-inducing MCD diet. However, an in vitro fluorescence microscopy assay demonstrates that MCD-medium is associated with increased ROS in murine hepatocytes.
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