Oxidative stress induced liver cell injury, secondary to obesity and diabetes, leads to the development of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD). Our long-term goal is to identify the mechanisms of mitochondrial induced liver cell damage that is critically necessary for the development of novel targeted molecular therapies. The specific hypothesis is the failure to adapt to the metabolic stress (reactive oxygen and nitrogen species- ROS/RNS) associated with hepatocellular injury, the hallmark of NASH, results from post- translational modifications of mitochondrial proteins leading to decreased synthesis and impaired function. Our hypothesis is based upon the observations that in subjects with NAFLD 1) ATP homeostasis is impaired, 2) mitochondrial structural abnormalities and mitochondrial DMA gene mutations/deletions have been described, and 3) 3-nitrotyrosine levels within hepatocytes is increased in NASH. These observations create the focus of this exploratory grant proposal which is to apply the novel 2-D blue native-electrophoresis proteomic technique to identify mitochondria and respiratory chain proteins and functional protein interactions within the liver cell in subjects with obesity, diabetes and NAFLD.
The specific aims are to: 1. Characterize the liver mitochondrial proteome in obese subjects with and without type 2 diabetes across the spectrum of NAFLD. Mitochondria will be isolated from liver biopsy specimens obtained from individuals undergoing bariatric surgery with type 2 diabetes and NASH. The mitochondrial proteome will be compared to age, sex and weight matched individuals without diabetes and normal or minimal fatty liver.
This aim will delineate mitochondrial proteins and identify defects in energy homeostasis enzymes that will help to elucidate the independent effects of diabetes on mitochondrial hepatic function. 2. Determine selected ROS/RNS-dependent post-translational modifications of the liver mitochondrial proteome in obese subjects with and without type 2 diabetes across the spectrum of NAFLD.
This aim i nvestigates the concept that increased ROS/RNS production leads to post- translational alterations in mitochondrial proteins resulting in defects in mitochondria respiratory complexes and/or dysfunctional associations between the complexes in NASH. The advantage of this approach is that future investigations will be capable to address protein: protein interactions and the pathogenic mechanisms of NASH to further pursue our long-term goal. ? ? ? ? ?
| Bailey, Shannon M; Andringa, Kelly K; Landar, Aimee et al. (2008) Proteomic approaches to identify and characterize alterations to the mitochondrial proteome in alcoholic liver disease. Methods Mol Biol 447:369-80 |
