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 by-products 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.

FIGURE 1.

Mitochondrial fatty acid handling and possible mechanisms of ROS generation during fatty acid oxidation. LCFA are activated on the mitochondrial outer membrane (MOM) to acyl-CoA and then taken up into mitochondria by the carnitine palmitoyltransferase (CPT) system, with conversion to acylcarnitine and then back to acyl-CoA. Electrons enter the ETC via β-oxidation and the tricarboxylic acid cycle (TCA) or via the ETF and ETF-QO. Possible sites of electron leakage and ROS formation are at complex I (at the flavin site or at the quinone binding site via RET) (13, 44, 54), at complex III (13, 51, 53), and from the ETF and ETF-QO (18). ROS generated at complex III can diffuse into the matrix and the intermembrane space (13, 51). If acyl-CoA supply exceeds the downstream catabolic capacity, acyl-CoA esters can be converted to acylcarnitines via carnitine acyltransferase (CrAT; with specificity for acetyl-CoA and short-chain acyl-CoA) and then exported from mitochondria via carnitine acylcarnitine transferase (CACT) (62). l-Carnitine supplementation increases acylcarnitine export (55–57). Because acyl-CoA esters can inhibit matrix ROS mitigating or detoxifying enzymes (22–25), acylcarnitine export may lower ROS during FAO. Rotenone (Rot) inhibits complex I. Antimycin A (Anti) binds cytochrome b to prevent electron flow to the ubiquinone binding site of complex III. Oligomycin (Oligo) inhibits the ATP synthase. MIM, mitochondrial inner membrane.

FIGURE 2.

H₂O₂ emission rate as a function of substrate concentration and type in skeletal muscle mitochondria. A, H₂O₂ emission rate with increasing supply of PCarn. Values are means ± S.E., n = 5 (n = 2 for 36 μm PCarn). *, **, and ***, p < 0.05, p < 0.01, and p < 0.001, respectively, significantly different from adjacent lower concentration; one-way analysis of variance (repeated measures), Bonferroni post-tests; analysis excludes 36 μm palmitoylcarnitine. B, raw fluorescence signals showing H₂O₂ emission with 18 μm palmitoylcarnitine but not with a saturating concentration of NADH-linked substrate (pyruvate/malate, 10/5 mm; glutamate/malate, 10/5 mm). H₂O₂ emission is indicated by an increase in the fluorescence signal. Inset, quantification (nmol H₂O₂/min/mg). Open bar, pyruvate/malate; closed bar, PCarn. Values are means ± S.E. (n = 5).

FIGURE 3.

Low energization of skeletal muscle mitochondria supplied with long-chain fatty acid substrate and relative insensitivity of H₂O₂ to Ψm. A, parallel determinations of Ψm and O₂ consumption in mitochondria respiring on 18 μm PCarn, 10/5 mm P/M, or 9 mm succinate (n = 2–5). Membrane potential was measured using a TPMP⁺-selective electrode. Bar graphs, statistical comparisons between PCarn and P/M. * and **, p < 0.03 and p < 0.01, respectively, paired t test. Values are means ± S.E. B, Ψm determinations by safranin fluorescence. Values are means ± S.E., expressed as a fraction of the 18 μm PCarn value (n = 3). C, Ψm and H₂O₂ emission rate in skeletal muscle mitochondria supplied with 18 μm PCarn or G/M (10/5 mm). FCCP was used to titrate the membrane potential (PCarn, 15 pm FCCP; G/M, 100 pm FCCP). Curve in C, the Ψm-H₂O₂ relationship with G/M was fit using the equation, y = 0.06267e^0.000183x (R² = 0.988). Arrows in C, values used for quantification in bar graphs. Bar graphs, average H₂O₂ emission rates at Ψm indicated by the arrows in C (n = 4). * and ***, p < 0.05 or p < 0.005, respectively, paired t test, low versus high Ψm. Values are means ± S.E. Numbers above bars summarize the approximate percentage decrease in H₂O₂ emission rate when going from lower to higher membrane potential.

FIGURE 4.

Topology and sites of ROS production in skeletal muscle mitochondria oxidizing long-chain fatty acid substrate. A, H₂O₂ emission rate of mitochondria oxidizing 18 μm PCarn in the absence or presence of added Cu/Zn-SOD (200 units/ml). B, as in A, with the addition of antimycin A (1 μm) to inhibit complex III. In A and B, the approximate percentage decrease increase in H₂O₂ emission rate with SOD addition is given. Increased H₂O₂ emission upon SOD addition indicates release of superoxide toward the intermembrane space. C, comparison of H₂O₂ emission rates in mitochondria supplied with PCarn (18 μm) or P/M (10/5 mm), in the presence of rotenone (1 μm) to inhibit complex I or antimycin plus SOD. The H₂O₂ emission rate was evaluated with a supply of succinate alone, which generates ROS by reverse electron transport to complex I. All values are means ± S.E. (n = 3–5). *, p < 0.03, paired t test, without SOD versus with SOD.

FIGURE 5.

l-Carnitine effects on bioenergetics and H₂O₂ emission rates of skeletal muscle and liver mitochondria. l-Carnitine (2 mm; Carn) was utilized to decrease levels of long-chain fatty acid catabolic intermediates within the mitochondrial matrix. Mitochondria (0.2 mg/ml) were supplied with 18 μm PCarn. A, in mitochondria supplied with PCarn plus [1-¹⁴C]palmitoylcarnitine, the [¹⁴C]acid-soluble product (¹⁴C-ASP) was used as a measure of LCFA catabolic intermediate levels within the matrix (pellet) and effluxed from mitochondria into the buffer (buffer). Above the bar in the muscle panel, a and b, p < 0.003 and p < 0.015 for buffer and pellet fractions, respectively, paired t test, without l-carnitine versus with l-carnitine. Above the bar in the liver panel, #, p = 0.08, paired t test, [¹⁴C]ASP was increased in the buffer fraction in four of four preparations. B, rate of ¹⁴CO₂ production. C–F, bioenergetic parameters (C and D) and H₂O₂ emission rates (E and F) were determined under the same conditions as in A and B, using only cold PCarn. In F, CDNB was used to deplete mitochondria of reduced glutathione. A–D, n = 4; E and F, n = 7–8. In all panels, values are means ± S.E. * and ***, p < 0.05 and p < 0.005, respectively, paired t test.

FIGURE 6.

Impact of NNT deletion on carnitine effects on H₂O₂ emission in skeletal mitochondria from C57Bl/6J and C57Bl/6 mice. Mitochondria were supplied with 18 μm palmitoylcarnitine. A, H₂O₂ emission rates. B, Ψm. C57Bl6/J mice harbor a five-exon deletion in the Nnt gene (see supplemental Fig. 5). Nnt encodes for NNT, which is thought to be largely responsible for NAD(P)H regeneration in mitochondria and thus to contribute to the maintenance of glutathione antioxidant defense. Values are means ± S.E., n = 3. ** and ***, p < 0.03 and p < 0.01, respectively, without carnitine versus with carnitine, two-way analysis of variance with Bonferroni post hoc tests.