Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates - PubMed (original) (raw)

Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates

J M Weinberg et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Kidney proximal tubule cells developed severe energy deficits during hypoxia/reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (DeltaPsi(m)). The energy deficit, respiratory inhibition, and loss of DeltaPsi(m) were strongly ameliorated by provision of alpha-ketoglutarate plus aspartate (alphaKG/ASP) supplements during either hypoxia or only during reoxygenation. Measurements of (13)C-labeled metabolites in [3-(13)C]aspartate-treated cells indicated the operation of anaerobic pathways of alphaKG/ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of alphaKG/ASP also mitigated the loss of DeltaPsi(m) that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F(1)F(0)-ATPase activity, had been responsible for maintenance of DeltaPsi(m) by the substrates. Thus, tubule cells subjected to hypoxia/reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and/or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain DeltaPsi(m) via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia.

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Figures

Figure 1

Energy deficit after H/R and its amelioration by αKG/ASP. (A) Cell ATP after 30, 45, 52.5, or 60 min of hypoxia followed by 60-min reoxygenation. C, control incubated under oxygenated conditions for 135 min. N ≥ 5 for each group. All H/R ATP values are significantly different from C and the preceding duration of hypoxia. (B) Minimal LDH release to the medium by cells depicted in A. (C) Basal and carbonyl cyanide-_n_-chlorophenylhydrazone (CCCP)-uncoupled respiration of intact tubules after 60-min hypoxia plus 60-min reoxygenation with either no extra substrate addition (NES) or 4 mM αKG/ASP during reoxygenation. N = 4; *, P < 0.05 vs. corresponding NES group; #, P < 0.05 vs. corresponding αKG/ASP group. (D) Respiration of digitonin-permeabilized tubules supported by 5 mM glutamate plus 5 mM malate (G/M), 5 mM succinate (SUCC), or 10 mM ascorbate plus 0.3 mM tetramethyl-_p_-phenylenediamine (A/T). Conditions and labeling are otherwise the same as for C. (E) Tubule cell ATP after 60-min hypoxia followed by 60-min reoxygenation with no extra substrate additions or αKG/ASP (4 mM each), αKG alone, or ASP alone during 60 min of hypoxia before reoxygenation or during only 60 min of reoxygenation (REOXY). N ≥ 8; *, P < 0.05 vs. corresponding no extra substrate group; #, P < 0.05 vs. corresponding αKG/ASP group. (F) ATP at the end of hypoxia before reoxygenation under the same conditions as E. Groups are labeled exactly as for E. Controls have lower ATP levels than controls in E because exogenous AMP supplements, which increase cell ATP (6), were provided only during reoxygenation.

Figure 2

Metabolic pathways for anaerobic generation of ATP promoted by αKG/ASP. (A) During hypoxia, αKG is metabolized to succinate (SUCC), forming GTP by substrate-level phosphorylation. GTP is transphosphorylated to ATP. Transamination of αKG to glutamate by aspartate provides oxalacetate (OAA). Conversion of OAA to malate (MAL) and fumarate (FUM) is linked to decarboxylation of αKG by the NAD-NADH redox couple (22). (B) Reduction of fumarate to succinate is coupled to oxidation of reduced ubiquinone (CoQred) generated via NADH by reducing equivalents from substrate (SUB) oxidation in the citric acid cycle (e.g., A) (21). This process anaerobically maintains electron flux and proton extrusion in complex I with ATP production by the mitochondrial inner membrane F1F0-ATPase. (Figure modified from ref. with permission.)

Figure 3

Isotopic enrichment studies with [3-13C]aspartate. Tubules were incubated for 60 min with 4 mM [3-13C]aspartate without or with 4 mM unlabeled αKG (−αKG, +αKG) during control incubation, hypoxia, or reoxygenation after 60-min hypoxia. A and B show 13C isotopic enrichments (Mole % Excess) into citrate and succinate, respectively. M+1 and M+2 are the proportions enriched at one and two carbons. C and D are mass measurements of total 13C citrate and succinate formed. N ≥ 3; *, P < 0.05 vs. corresponding control; #, P < 0.05 vs. corresponding −αKG group.

Figure 4

Mitochondrial ultrastructural changes. (a) Control. (b) Sixty-minute hypoxia. (c and d) Sixty-minute hypoxia followed by 60-min reoxygenation. Arrowhead, mitochondrion with high-amplitude swelling. (×24,100.)

Figure 5

Effect of αKG/ASP on mitochondrial ultrastructure. (a) Sixty-minute hypoxia with 4 mM αKG/ASP. (b) Sixty-minute hypoxia followed by 60-min reoxygenation with 4 mM αKG/ASP during hypoxia and reoxygenation. (×24,100.)

Figure 6

Mitochondrial uptake of JC-1. (a) Control. (b) Sixty-minute hypoxia plus 60-min reoxygenation. (c) Sixty-minute hypoxia plus 60-min reoxygenation with 4 mM αKG/ASP during reoxygenation. _a_– c show JC-1-loaded tubules viewed simultaneously by confocal microscopy for green fluorescence (488-nm excitation, 522-nm emission, Left) and red fluorescence (568-nm excitation, 585-nm emission, Right). (Bar = 10 μm.)

Figure 7

Quantitative changes of JC-1 fluorescence. Typical scans (488-nm excitation and 510- to 625-nm emission) of JC-1-loaded tubules under control conditions, in the presence of 5 μM FCCP plus 5 mM glycine for 15 min, or for 60-min hypoxia plus 60-min reoxygenation with either no extra substrates (NES) or 4 mM αKG/ASP during reoxygenation. Values next to the legends (Inset) are the ratios of the signal intensities at 590 and 530 nm for groups studied under each condition, N ≥ 8. *, P < 0.05 vs. corresponding NES group; #, P < 0.05 vs. corresponding αKG/ASP group.

Figure 8

Effect of αKG/ASP on ΔΨm during hypoxia. (A) JC-1 fluorescence ratios (590/530 nm) after hypoxia for 60 min at pH 6.9 in the presence of 20 μM antimycin A and 2 μM 5-_n_-nonyl-6-hydroxy-4,7-dioxobenzothiazol (H+N+A) with no extra substrate (NES) or 4 mM αKG/ASP. In separate groups, the medium additionally contained 15 μM oligomycin (+O), 10 μM rotenone (+R), or rotenone plus oligomycin (+R+O). N ≥ 4; *, P < 0.05 vs. corresponding NES group. (B) ATP levels for the experiments depicted in A.

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Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates - PubMed (original) (raw)