Manipulating /Preventing Mitochondrial Oxidative Stress

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DESCRIPTION (provided by applicant): Hyperglycemia is a major independent risk factor for diabetic macrovascular disease. The consequences of exposure of endothelial cells to hyperglycemia are well established. However, little is known about how adipocytes respond to both acute as well as chronic exposure to physiological levels of hyperglycemia. Our recent observations have highlighted dramatic effects of increased ROS levels in adipocytes. We have established that hyperglycemia leads to increased mitochondrial production of ROS levels. This leads to a general induction of the inflammatory response, ROS-induced DNA damage and overall insulin resistance in adipocytes. Importantly, we have demonstrated that we can manipulate inflammation and insulin resistance independent of hyperglycemia through direct manipulation of mitochondrial ROS generation by altering the expression levels of the mitochondrial dicarboxylate transporter, mDIC. This transporter acts as a rate-limiting factor for substrate availability for the electron transport chain in adipocytes. Furthermore, we have found strong evidence indicating that PPARgamma agonists exert rapid transcription-independent effects on mitochondria in adipocytes that lead to a dose-dependent reduction of ROS levels. This may occur through a direct interaction with a 17kD mitochondrial membrane protein called mitoNEET. Here, we aim to determine the role of the mDIC in the control of ROS production in adipocytes, characterize the effects of PPARgamma agonists on mitochondrial membrane potential, and determine whether mitoNEET is a critical player in the effects of TZDs on ROS in adipocytes. Specifically, we aim to: SA 1) Demonstrate that mDIC is critically involved in ROS generation by genetically removing the mDIC gene from mouse adipocytes ("adipocyte-specific null"), reducing the levels of mDIC in vivo as well as overexpressing it in adipocytes. SA 2) Define the direct effects of PPARgamma agonists on mitochondria in adipocytes. SA 3) Analyze the contributions of mitoNEET as a PPARgamma agonist target in adipocytes. The increased presence of ROS in adipocytes has a profound impact on cellular insulin sensitivity. ROS-induced damage in these very long-lived cells accumulates over time and displays a "hyperglycemic memory" in that ROS-induced damage is not readily reversible. This proposal aims to highlight the contribution of two specific candidate molecules, mDIC and mitoNEET, towards the generation of ROS in adipocytes, thereby providing two new promising therapeutic targets.