1a. Objectives (from AD-416)
1. Determine the extent to which dietary antioxidants alter obesity-induced and/or exercise-induced changes in mitochondrial function and insulin sensitivity. Sub-objective 1A. Determine the influence of anti-oxidant supplementation on changes in insulin sensitivity induced in the rat by high dietary fat and exercise. Sub-objective 1B. Determine the degree to which anti-oxidant supplementation alters exercise-induced changes in insulin sensitivity and mitochondrial function responses of overweight/obese individuals. 2. Identify sites and causes of obesity-induced and exercise-induced oxidative stress. Sub-objective 2A. Determine the effects of obesity and exercise on the temporal and cellular activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf-2)/Anti-oxidant Response Element pathway. Sub-objective 2B. Identify and characterize obesity-induced and exercise-induced oxidative changes to insulin signaling pathway proteins. 3. Identify, characterize and compare sites of obesity-induced versus exercise-induced mitochondrial respiratory changes. Sub-objective 3A. Determine the degree to which anti-oxidant supplementation blunts exercised-induced and obesity-induced changes in mitochondria.
1b. Approach (from AD-416)
In order to complete the objectives of this proposal, we will utilize a combination of studies in humans, rodents that examine physiologic, metabolomic, genetic, and proteomic endpoints. In Objective 1, we will perform studies in humans and rodents to determine how antioxidant (vitamin E and vitamin C) supplementation affects insulin responses to exercise and obesity. The study in humans will involve analysis of exercise adaptation and insulin responses in previously untrained individuals and if antioxidant supplementation either enhances or negates these adaptations. Rodent studies will further examine molecular mechanisms underlying these adaptations. In Objective 2, we will determine the extent to which obesity, exercise, and anti-oxidant supplementation alter redox balance in animals and specific cells and to identify specific proteins whose thiol redox status is altered in obesity, exercise, and anti-oxidant supplementation. These studies will utilize transgenic mouse models and proteomic approaches. In Objective 3, we will determine the extent to which obesity, exercise, and anti-oxidant supplementation alter mitochondrial function. These studies will utilize rat models of exercise and obesity. Whole tissue and isolated mitochondria will be studied for changes in total mitochondrial content, mitochondrial gene expression, and respiration, and mitochondrial enzyme activities.
3. Progress Report
This is the annual report for the new OSQR-approved Project 5450-51000-048-00D that replaces Project 5450-51000-042-00D. (See separate annual report terminating that project.) One focus of this project is to determine the extent to which anti-oxidant supplementation alters obesogenic diabetes as well as the positive, insulin-sensitizing and mitochondrial effects of exercise. Another focus is to determine how oxidative stress signaling may be a positive and/or negative response to obesity and exercise depending on the target tissues and proteins. During this time, we have been working on Objective 1 and Objective 2. For Obj. 1, we have performed initial experiments demonstrating the utility of the obese-prone Sprague-Dawley rat as a model for obesity-induced insulin resistance. Our next step is to induce obesity in these animals in the presence of vitamin E and vitamin C and to determine the extent to which this supplementation prevents insulin resistance. We are also developing the protocol manual for our clinical study evaluating the effects of anti-oxidants upon exercise adaptation in humans. Using tissues from obese versus lean animals we demonstrated that the activity of the oxidant response protein methionine sulfoxide reductase is reduced in the adipose of obese animals and that levels of methionine sulfoxide modified proteins are also reduced. These data are important since methionine sulfoxidation regulates intracellular signaling and thus opens an avenue identifying changes in targets of oxidative stress and how they are altered by obesity. This work will be submitted for publication. For Obj.2, we are waiting to start the transgenic colony proposed owing to infection of the mice at the provider’s facility. We will have to regenerate the line from the infected animals via a third party (e.g. Jackson Labs) that specializes in this procedure. In the mean time, we are pursuing an alternate strategy in which we are using transgenic mice lacking the anti-oxidant response pathway to determine whether they are unable to effectively adapt to exercise. We have begun preliminary proteomic studies to identify sites of oxidative damage to phosphatases (PTEN and PTP1B) involved in insulin signaling.