Preliminary Data Monitoring In Vivo Sources of Reactive Oxygen Species Production in Human Obesity

Abstract

Introduction Excessive reactive oxygen species (ROS) production is a prominent feature of obesity-related conditions including impairments in endothelial function. NADPH oxidases (Nox) and mitochondria are major sources of ROS production within the vasculature. However, studies involving direct measurement of in vivo ROS production are limited in humans. Thus, the relative contributions of Nox- and mitochondrial-derived ROS in human obesity remain to be fully characterized. The objective of this study was to determine the predominant sources of in vivo ROS production in the skeletal muscle microvasculature of individuals with obesity. We hypothesize that both Nox- and mitochondria-dependent ROS production are upregulated in human obesity. Methods Two sedentary men with Class II and above obesity (body mass index ≥ 35 kg/m2) and the Metabolic Syndrome were studied. Microdialysis was used for measurements of in vivo skeletal muscle ROS production. Microdialysis probes were perfused with saline containing Amplex Ultrared, horseradish peroxidase and superoxide dismutase to measure local hydrogen peroxide and superoxide concentrations. To assess ROS production from distinct sources, microdialysis probes were perfused with the following: apocynin (non-specific Nox inhibitor), MitoTEMPO (mitochondrial-ROS inhibitor), and GKT 137831 (specific Nox 4 inhibitor). Results The concentrations of hydrogen peroxide and superoxide decreased upon addition of apocynin to microdialysis probes (mean ± SEM; hydrogen peroxide: 1.18 ± 0.58 to 0.76 ± 0.01 μM; superoxide: 1.47 ± 0.46 to 1.10 ± 0.62 μM). MitoTEMPO perfusion elicited an increase in hydrogen peroxide (1.12 ± 0.76 to 1.51 ± 1.08 μM) and decrease in superoxide (1.47 ± 0.46 to 0.78 ± 0.10 μM). The combination of apocynin plus MitoTEMPO resulted in concentrations of hydrogen peroxide (0.22 ± 0.10 μM) and superoxide (0.23 ± 0.06 μM) that were 69.7% and 79.1% lower than apocynin alone. In addition, the hydrogen peroxide (0.56 ± 0.25 μM) and superoxide (0.43 ± 0.18 μM) concentrations with co-perfusion of apocynin and GKT were 26.3% and 60.9% lower than with apocynin alone. Conclusions Our preliminary data suggest that mitochondria and Nox together are significant sources of ROS production within the skeletal muscle microvasculature of obese individuals. The local inhibition of Nox- and mitochondrial-derived ROS resulted in marked decreases in extracellular ROS levels within the skeletal muscle microvasculature. Although the small sample size currently limits our interpretation of the results, this study is anticipated to become the first to report on distinct sources of in vivo ROS production in human obesity.