Background Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Δψm), which is usually generated by the respiratory chain complexes. We investigated the source of Δψm in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this Δψm for the generation of ATP. Methods and Principal Findings Individual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but Δψm was still decreased by inhibition of complex III, confirming the role of the respiratory chain in maintaining Δψm. Complex V did not maintain Δψm by consumption of ATP, as has previously been suggested for eosinophils. We show that complex III in neutrophil mitochondria can receive electrons from glycolysis via the glycerol-3-phosphate shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were lacking in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercomplex organisation while gaining increased aerobic glycolysis, just like neutrophils. Conclusions We show that neutrophils can maintain Δψm via the glycerol-3-phosphate shuttle, whereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors.
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