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 1H and two-dimensional 1H/13C heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. These techniques proved far more sensitive than conventional 31P 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.

A Mitochondrial-Targeted Coenzyme Q Analog Prevents Weight Gain and Ameliorates Hepatic Dysfunction in High-Fat–Fed Mice

“…This technique enabled bioenergetic studies to be carried out over respiratory states ranging from state 4 (no added ADP) to state 3 (ADP added in different amounts). For this method to be effective, the membrane potential should decrease in stepwise fashion to plateau levels with each incremental addition of ADP (or ATP), which is the case, as we have shown in the past for muscle (Yu et al, 2013), liver (Yu et al, 2014), and heart (Yu et al, 2014) mitochondria.…”

“…The addition of substrates to respiratory buffer without mitochondria did not affect fluorescence. Amplex Red does not interfere with ATP production or with NMR detection of 2DOGP (Yu et al, 2013).…”

“…NMR spectra were collected at 37 o C on a Bruker Avance II 500 MHz NMR spectrometer. Mitochondrial samples were studied by acquiring two-dimensional 1 H/ 13 C-HSQC NMR spectra using 13 C-labeled 2DOG at C6-position ([6-13 C]2DOG) as we recently described (Yu et al, 2013). The amount of 2DOG and 2DOGP present in the NMR samples were quantitatively measured using the peak intensities of the assigned resonances of these compounds.…”

“…We used a novel method that we recently described (Yu et al, 2013) to carry out bioenergetic studies of isolated mitochondria under conditions of clamped ADP and membrane potential (DC). Studies were carried out in the presence of excess 2-deoxyglucose (2DOG) and hexokinase and varying amounts of added ADP or ATP.…”

Mitochondria and Oxidative Stress in Diabetes

“…ROS production viewed independent of e − transport is analogous to motor vehicle heat generation viewed independent of the speed of the vehicle. In a recent manuscript, we also reported an increase in superoxide generation per unit ATP produced in STZdiabetic muscle mitochondria [68].…”