Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation

Abstract

Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and byproducts of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels. Reactive oxygen species (ROS)2 are generated in mitochondria as a normal byproduct of aerobic metabolism; 0.2-2% of O 2 consumption is estimated to be lost as superoxide under normal conditions (1, 2). Within the mitochondrial matrix, a suite of enzymes manages the ROS load by converting ROS to less toxic species or by mitigating their formation. Nevertheless, mitochondria are significant sources of cellular ROS, and oxidative stress is a hallmark of various physiological and pathological states, including exercise, insulin resistance, and atherosclerosis (3-10).Inhibitor studies in mitochondria utilizing NADH-or FADH 2 -linked substrates suggest that complexes I and III of the electron transport chain (ETC) are predominant sites of superoxide production (11-13). Whether this is the case under physiologic conditions is not well appreciated. ROS generation by the ETC is critically dependent upon ETC redox state, such that ROS production is low until the inner membrane is significantly polarized and then rises steeply with small increments in membrane potential (14). However, a membrane potential-independent component of ROS production has been observed in brain mitochondria (15)(16)(17). Indeed, electrons may escape from sites other than the respiratory complexes, such as from the electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETF-QO) (18) or the ␣-ket...

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

“…A stronger candidate is the ETF system (ETF and ETFQOR) because it has been implicated in superoxide/H 2 O 2 production in skeletal muscle (54). 3 Several lines of evidence do not support a role for it in what we define as mGPDH-specific superoxide production.…”

Lipotoxicity in the liver

“…Palmitate-induced oxidative stress has been reported to contribute to insulin resistance in H4IIEC3 rat hepatocytes [40] . Fatty acid accumulation stimulates ROS generation in the liver presumably due to enhanced β-oxidation and to the consequent electron overflow in the mitochondrial electron transfer chain [41] ( Figure 2B). However, a decrease in mitochondrial quinone pool and a related inhibition of mitochondrial oxidative metabolism were also suggested to underlie the increased mitochondrial ROS production in high fat diet [42] .…”

Evaluation of Mitochondrial Functions and Dysfunctions in Muscle Biopsy Samples

“…The latter situation is suggested to be the most physiologically relevant ROS production in mammals (Miwa & Brand, 2003). Recent work from Seifert et al (2010) showed that LCFA β-oxidation is associated with enhanced ROS production, through a mechanism involving complex III, the electron transfer flavoprotein (ETF) and ETF-oxidoreductase.…”

“…Similarly, a small decrease in membrane potential deeply reduces ROS production (Votyakova & Reynolds, 2001). Therefore mild uncoupling resulting from a small decrease in membrane potential is considered as a natural antioxidant process (Seifert et al, 2010;Skulachev, 1997). However, not all sites of ROS production are sensitive to membrane potential.…”

“…However, not all sites of ROS production are sensitive to membrane potential. Indeed, Seifert et al (2010) found that the ROS produced during LCFA β-oxidation and involving complex III, ETF and ETF-oxidoreductase, was relatively insensitive to membrane potential changes. In addition, Miwa and Brand reported that the ROS produced at the cytosolic side of complex I through glycerol-3-phosphate dehydrogenase, which donates electrons to the electron carrier Q, is insensitive to membrane potential (2003).…”