The goal of the work proposed in this application is to decrease fat stores by increasing oxidation. The hypothesis underlying this approach is that preventing inhibition of respiration induced by ROS will lead to increased oxidation and decreased fat storage. Adipocyte respiration above basal is inhibited. However, scavengers of reactive oxygen species (ROS), pyruvate or N-acetyl cysteine, can restore O2 consumption in rat adipocytes and decrease fat stores in human adipocytes. This was observed only in intact cells and not in isolated mitochondria suggesting that ROS production required the high fat environment of the intact cell. We have developed a novel long-lasting preparation of immobilized cultured adipocytes that permits repeated measurement of O2 consumption and image analysis of fluorescent molecules. To test our hypothesis we will use isolated rat adipocytes and mitochondria and differentiated human preadipocytes to perform the following Aims:
Aim 1 is to determine if ROS removal stimulates respiration and decreases TG stores in rat and human adipocytes. The prediction will be tested that there is a relationship between ROS and TG stores such that decreasing ROS through over-expression of superoxide dismutase, peroxiredoxin or glutathione peroxidase will decrease TG stores. We will simultaneously measure mitochondria! membrane potential (&W)and respiration to assess coupling.
Aim 2 is to identify the site in the respiratory chain that is inhibited by ROS. To learn the identity of the site, we will measure O2 consumption in adipocytes from substrates that enter the respiratory chain through Complexes I, III or IV. Relief of inhibition will be indicated by increased respiration.
Aim 3 is to determine whether O2"""""""", H2O2, NO or other ROS mediate inhibited respiration. Using submitochondrial particles derived from adipocytes we will identify the inhibitory ROS species and their specific site of action. Flux will be measured in the presence of ROSand site-specific substrates.
Aim 4 will determine the mechanism of low adipocyte apoptosis and turnover amid evidence of strong ROSaction in mitochondria. We will map mitochondrial networking in adipocytes using photoactivation and test the effect of network alteration through gene knockdown. We anticipate that uncoupling between ROS and apoptosis is an essential component of adipocyte FFA handling. The anticipated outcome of these experiments is identification of the mechanism responsible for the unique inhibition of adipocyte respiration by ROS and ways to overcome this inhibition. Respiration in adipocytes has been little investigated, due in part, to the misconception that it is not important. These experiments could reverse the thrifty phenotype and possibly lend insight into the thrifty genotype and its role in the development of obesity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056690-10
Application #
7766908
Study Section
Special Emphasis Panel (ZRG1-EMNR-B (90))
Program Officer
Haft, Carol R
Project Start
1999-12-01
Project End
2011-03-31
Budget Start
2010-02-01
Budget End
2011-03-31
Support Year
10
Fiscal Year
2010
Total Cost
$378,842
Indirect Cost
Name
Boston Medical Center
Department
Type
DUNS #
005492160
City
Boston
State
MA
Country
United States
Zip Code
02118
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