Diet-sensitive sources of reactive oxygen species in liver mitochondria: role of very long chain acyl-CoA dehydrogenases

Abstract

High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis is accompanied by enhanced generation of reactive oxygen species (ROS), which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is also a source of ROS production in mitochondria of HFD animals. This production is stimulated by the lack of NAD(+). Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS.

Figure 2. H₂O₂ release from liver homogenate fractions.

Liver homogenates from control and HFD animals were prepared as described in Materials and Methods and incubated in buffer containing 150 mM KCl, 2 mM MgCl₂, 1 mM EGTA, 1 mM KH₂PO₄, 10 mM Hepes, 0.1% BSA, pH 7.4. 5 μM Amplex Red and 1 U/mL HRP were present to measure H₂O₂. (A) Carbonylated proteins were detected as described in Materials and Methods; (B) H₂O₂ release from the total (600 g) and mitochondrially-removed (7000 g) fractions; (C) Mitochondrial fraction incubated in the presence of 2 mM succinate; (D) Mitochondrial fraction incubated in the presence of 50 μM palmitoyl-CoA (palm-CoA) and 50 μM malonyl-CoA (mal), where indicated; (E) Percoll-purified mitochondrial fractions incubated in the presence of 50 μM palmitoyl-CoA; (F) Mitochondrial fraction incubated in the presence of 50 μM palmitoyl-carnitine (palm-carn) and 50 μM malonyl-CoA (mal); *, p < 0.05 versus control, ^#, p < 0.05 versus HFD in the absence of malolyl-CoA, n = 6 per group.

Figure 3. Mitochondrial respiration and oxidative phosphorylation are unchanged by HFD.

(A) Mitochondria (0.5 mg protein. mL^-1) were incubated in 120 mM sucrose, 65 mM KCl, 2 mM MgCl₂, 1 mM KH₂PO₄, 1 mM EGTA, 10 mM Hepes, 0.1% BSA, pH 7.4. Succinate (2 mM), 1 μg/mL oligomycin, 1 mM ADP and 1 μM CCCP were added. Oxygen consumption was measured as described in Materials and Methods. (B) Respiratory control (RCR) and ADP/O ratios were calculated under the experimental conditions of Panel A. (C) Mitochondrial inner membrane potentials were measured under the conditions of Panel A, as described in Materials and Methods, n = 6 per group.

Figure 4. Mitochondrial respiration supported by palmitoyl-carnitine is enhanced in HFD.

Mitochondria were incubated under the conditions described for Figure 3, substituting succinate for 50 μM palmitoyl-carnitine. Malonyl-CoA (mal, 50 μM) was present where indicated. *, p < 0.05 versus control, ^#, p < 0.05 versus HFD in the absence of malonyl-CoA, n = 6 per group.

Figure 5. HFD increases the expression of acyl-CoA dehydrogenases isoforms and electron transfer flavoprotein (ETF).

Mitochondrial acyl-CoA dehydrogenases and ETF subunits A and B (A-B), were quantified by western blotting as described in Materials and Methods. (A) Representative blots (note that VLCAD has two isoforms), (B) averages ± SEM of densitometries. Where indicated *, p < 0.05 versus control, n = 6 per group.

Figure 6. HFD increases ROS release from VLCAD.

(A) Frozen and defrosted mitochondria (0.5 mg protein. mL^-1) were incubated in 150 mM KCl, 2 mM MgCl₂, 1 mM EGTA, 1 mM KH₂PO₄, 10 mM Hepes, 0.1% BSA, pH 7.4. Alamethicin (0.5 μg/mL), 0.5 ng/mL antimycin A, 1 µM rotenone and 50 µM NAD⁺ were added to the incubation media, and NADH fluorescence was monitored as described in Materials and Methods in the presence of 50 μM butyryl-CoA, octanoyl-CoA or palmitoyl-CoA, where indicated. (B) Frozen and defrosted mitochondria were incubated in the same media as Panel A, in the presence of 0.5 μg/mL alamethicin, 5 μM Amplex Red and 1 U/mL HRP. H₂O₂ release was measured as described in Materials and Methods. (C) Fresh mitochondria were incubated under the conditions of Figure 3, substituting succinate for 50 μM butyryl-CoA, octanoyl-CoA or palmitoyl-CoA, as shown. Where indicated, 50 µM malonyl-CoA (mal) was present. *, p < 0.05 versus control, ^#, p < 0.05 versus HFD in absence of malonyl-CoA, n = 6 per group.

Figure 7. NAD⁺ prevents H₂O₂ release by VLCAD.

Frozen and defrosted mitochondria (0.2 mg protein. mL^-1) were incubated under the conditions of Figure 6B, in the presence of varying NADH (Panel A) or NAD⁺ (Panel B) concentrations, as indicated. H₂O₂ release was monitored as described in Materials and Methods, n = 6 per group.

Figure 1. HFD leads to enhanced micro vesicular lipid droplets in livers.

Panels A and B: Sections were stained with Hematoxylin and Eosin (H and E). Panels C and D: Oil red O staining shows and HFD livers have more areas marked with this dye in regions near the lumen of vessels. “L” indicates the lumen, and a sample hepatocyte is marked with an asterisk. Images are representative of serial sections from 6 livers per group.