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

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 ΔΨ_m 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 ΔΨ_m, 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).