title: Mitochondrial function in diabetes: novel methodology and new insight.
abstract: Interpreting mitochondrial function as affected by comparative physiologic conditions is confounding because individual functional parameters are interdependent. Here, we studied muscle mitochondrial function in insulin-deficient diabetes using a novel, highly sensitive, and specific method to quantify ATP production simultaneously with reactive oxygen species (ROS) at clamped levels of inner mitochondrial membrane potential (ΔΨ), enabling more detailed study. We used a 2-deoxyglucose (2DOG) energy clamp to set ΔΨ at fixed levels and to quantify ATP production as 2DOG conversion to 2DOG-phosphate measured by one-dimensional (1)H and two-dimensional (1)H/(13)C heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. These techniques proved far more sensitive than conventional (31)P nuclear magnetic resonance and allowed high-throughput study of small mitochondrial isolates. Over conditions ranging from state 4 to state 3 respiration, ATP production was lower and ROS per unit of ATP generated was greater in mitochondria isolated from diabetic muscle. Moreover, ROS began to increase at a lower threshold for inner membrane potential in diabetic mitochondria. Further, ATP production in diabetic mitochondria is limited not only by respiration but also by limited capacity to use ΔΨ for ATP synthesis. In summary, we describe novel methodology for measuring ATP and provide new mechanistic insight into the dysregulation of ATP production and ROS in mitochondria of insulin-deficient rodents.
authors: NMR Core Facility and Department of Biochemistry, University of Iowa, Iowa City, Iowa, USA.
authors: Liping
authors: L
authors: Yu
authors: Brian D
authors: BD
authors: Fink
authors: Judith A
authors: JA
authors: Herlein
authors: William I
authors: WI
authors: Sivitz
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003920:Diabetes Mellitus / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide