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. 2018 May 25;293(21):8032-8047.
doi: 10.1074/jbc.RA118.003200. Epub 2018 Apr 6.

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift

Jane A Armstrong  1 , Nicole J Cash  2 , Yulin Ouyang  2 , Jack C Morton  2 , Michael Chvanov  2 , Diane Latawiec  1 , Muhammad Awais  1 , Alexei V Tepikin  2 , Robert Sutton  1 , David N Criddle  3
Affiliations

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift

Jane A Armstrong et al. J Biol Chem. .

Abstract

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1-10 μm), whereas higher levels (0.5-1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 μm to 1 mm H2O2), with maximal effects at 500 μm H2O2 H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 μm H2O2 However, higher H2O2 levels (≥50 μm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation.

Keywords: Acute Pancreatitis; Seahorse; antioxidant; apoptosis; bioenergetics; cyclophilin D; mitochondrial permeability transition (MPT); necrosis (necrotic death); oxidative stress; pancreas; reactive oxygen species (ROS).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Effects of H2O2 on intracellular ROS levels and cell death. Concentration-dependent effects of H2O2 (1 μm to 1 mm) on intracellular ROS levels (A, chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), apoptosis (B, panel i, caspase-3/7, green), and necrosis (B, panel ii, propidium iodide) in isolated murine pancreatic acinar cells measured over a 13-h period (H2O2 was applied at time 0). The changes are normalized increases in fluorescence from the baseline (F/F0) and expressed as the means ± S.E. (n = 6). Significant differences from the control are shown as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
Figure 2.
Figure 2.
Effects of H2O2 on redox ratio and mitochondrial membrane potential. A, panel i, typical images showing the transmitted light (left panel) and mitochondrial localization of NADH and FAD+ autofluorescence in a pancreatic acinar cell, measured simultaneously using confocal microscopy. Panel ii, concentration-dependent effects of H2O2 (10–300 μm) and CCCP on NADH and FAD+ levels, expressed as normalized values from control (F/F0). Panel iii, effects of H2O2 (10 μm to 1 mm) on the redox ratio (NADH/FAD+) expressed as percentages of basal values; CCCP applied to show a maximal effect. B, effects of H2O2 on mitochondrial membrane potential (ΔΨm), expressed as the percentages of decrease of TMRM fluorescence (averages of ≥56 cells from >4 animals). The data have been normalized to the initial fluorescence at t = 0 (F/F0). All data shown are the means ± S.E. Significant differences from the control are shown as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.
Figure 3.
Figure 3.
Effects of H2O2 on mitochondrial bioenergetics. A and B, the effects of H2O2 (●) at 30 μm (A) and 100 μm (B) on the OCR in isolated pancreatic acinar cells compared with controls without H2O2 addition (□). C–G, a respiratory function “stress” test was carried out using sequential addition of oligomycin (Oligo, 1 μg/ml), FCCP (0.3 μm), and rotenone (Rot)/antimycin A (Anti A) combined (1 μm) injected sequentially. Acute effects of H2O2 (10–100 μm) on baseline changes of OCR (C) and ECAR (D) after 5 min (black) and 30 min (gray), ATP turnover capacity (E), spare respiratory capacity (F), and proton leak (G). The data are shown as the means ± S.E. (n = 3). Significant differences from the control are shown as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
Figure 4.
Figure 4.
Effects of menadione on mitochondrial bioenergetics. A and B, the effects of menadione (●) at 5 μm (A) and 10 μm (B) on the oxygen consumption rate (OCR) in isolated pancreatic acinar cells compared with controls without menadione addition (□). A respiratory function “stress” test was carried out using sequential addition of oligomycin (Oligo, 1 μg/ml), FCCP (0.3 μm), and rotenone (Rot)/antimycin A (Anti A) combined (1 μm) injected sequentially. C–G, acute effects of menadione (5–30 μm) on baseline changes of OCR (C) and ECAR (D) after 5 min (black) and 30 min (gray), ATP turnover capacity (E), spare respiratory capacity (F), and proton leak (G). The data are shown as the means ± S.E. (n = 8). Significant differences are shown as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with control; and $, p < 0.05; $$, p < 0.01; and $$$, p < 0.001 compared with the 5-min time point.
Figure 5.
Figure 5.
Effects of H2O2 and menadione on ATP levels and cell viability. A and B, the effects of H2O2 (10–100 μm) and menadione (5–50 μm) on ATP levels measured via luciferase assay (A) and cell viability (lactate dehydrogenase levels (LDH)) (B). Oligomycin (Oligo, 1 μg/ml) was added to show a maximal effect in A, panel ii. The data are shown as the percentages of viability, expressed as the means ± S.E. (n = 3). Significant differences from the control are shown as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
Figure 6.
Figure 6.
Effects of N-acetylcysteine on menadione-induced mitochondrial bioenergetic inhibition. A–D, the effects of NAC (250 μm) on menadione-induced (MEN, 10 μm) changes of the OCR in isolated pancreatic acinar cells after 5 min (A) and after 30 min (B), ATP turnover capacity (C), and spare respiratory capacity (D, n = 3). E, the effects of NAC (250 μm) on menadione-induced (10 and 30 μm) reductions of ATP levels measured via luciferase assay. The data are shown as the means ± S.E. (n = 4). Significant differences from the control are shown as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with control; and †, p < 0.05; ††, p < 0.01; and †††, p < 0.001 compared with menadione alone.
Figure 7.
Figure 7.
Effects of NAC on H2O2-induced cell death. The effects of 250 μm NAC are shown on the concentration-dependent actions of H2O2 (10 and 500 μm) on apoptosis (A, caspase-3/7, green) and necrosis (B, propidium iodide) in isolated murine pancreatic acinar cells measured at 13 h (H2O2 was applied at time 0). The changes are normalized increases of fluorescence from the baseline (F/F0) and expressed as the means ± S.E. (n = 3). Significant differences from the control are shown as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
Figure 8.
Figure 8.
Effects of cyclophilin D knockout (Ppif−/−) and pharmacological inhibition on H2O2-induced mitochondrial depolarization and reduction of NADH. A–D, the effects of 50 μm (A and B) and 500 μm (C and D) H2O2 on mitochondrial membrane potential (ΔΨm) and NADH autofluorescence in pancreatic acinar cells isolated from Ppif−/− (gray) and WT (C57Bl6, black) mice. The effects of cyclosporine A (CsA) treatment on changes in WT to H2O2 are also shown. Changes are expressed as normalized decreases of TMRM fluorescence and falls of NADH from basal levels (F/F0); the mitochondrial uncoupler CCCP (10 μm) was applied to show maximal depolarization. E and F, the concentration-dependent rates of fall of TMRM fluorescence and NADH autofluorescence in response to H2O2 under the various conditions are displayed. All data are shown as the means ± S.E. (averages of ≥80 cells from >4 animals).
Figure 9.
Figure 9.
Effects of cyclophilin D knockout (Ppif−/−) on H2O2-induced changes of mitochondrial bioenergetics. The effects of H2O2 (30 and 100 μm) on the OCR in pancreatic acinar cells isolated from Ppif−/− (gray) and WT (C57Bl6, black) mice after 5 min (A) and after 30 min (B), ATP turnover capacity (C), and spare respiratory capacity (D). The data are shown as the means ± S.E. (n = 3).
Figure 10.
Figure 10.
Effects of cyclophilin D knockout (Ppif−/−) and pharmacological inhibition on H2O2-induced cell death. A and B, the effects of H2O2 (10 and 100 μm) on apoptosis (A, caspase 3/7, green) and necrosis (B, propidium iodide) in pancreatic acinar cells isolated from Ppif−/− (gray) and WT (C57Bl6, black) mice (panel i) and in WT (black) and CsA-treated WT (gray) (panel ii). The data were normalized to the initial fluorescence at time 0 (F/F0) and expressed as the means ± S.E. (n = 6).
Figure 11.
Figure 11.
Effects of cyclophilin D knockout (Ppif−/−) and pharmacological inhibition on menadione-induced mitochondrial depolarization, reduction of NADH, and pancreatic acinar cell death. A and B, the effects of menadione (MEN, 30 μm) on mitochondrial membrane potential (A, ΔΨm) and NADH autofluorescence (B) in pancreatic acinar cells isolated from Ppif−/− (gray) and WT (C57Bl6, black), and in WT with CsA treatment. The changes are expressed as normalized decreases of TMRM fluorescence and falls of NADH from basal levels (F/F0); the mitochondrial uncoupler CCCP (10 μm) was applied to show maximal depolarization. C and D, the effects of menadione (30 and 500 μm) on apoptosis (C, caspase 3/7, green) and necrosis (D, propidium iodide) in pancreatic acinar cells isolated from Ppif−/− (gray) and WT (9C57Bl6, black) mice. All data are shown as the means ± S.E. (n ≥ 3).
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