Mitochondrial Membrane Potential in Human Neutrophils Is Maintained by Complex III Activity in the Absence of Supercomplex Organisation

textabstractBackground: Neutrophils depend mainly on glycolysis for their enegry provision. Their mitochondria maintain a membrace potential (ΔΨm), which is usually generated by the repiratory 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 byinhibition 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 shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were ladding in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercimplex organisation while gaining increased aerobic glycolysis, just like neutrophils. Conclusions: We show that neutrophils can maintain ΔΨm via the glycerol-3-phosphate shuttle, wereby 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

Mitochondrial Lactate Dehydrogenase Is Involved in Oxidative-Energy Metabolism in Human Astrocytoma Cells (CCF-STTG1)

Lactate has long been regarded as an end product of anaerobic energy production and its fate in cerebral metabolism has not been precisely delineated. In this report, we demonstrate, for the first time, the ability of a human astrocytic cell line (CCF-STTG1) to consume lactate and to generate ATP via oxidative phosphorylation. 13C-NMR and HPLC analyses aided in the identification of tricarboxylic acid (TCA) cyle metabolites and ATP in the astrocytic mitochondria incubated with lactate. Oxamate, an inhibitor of lactate dehydrogenase (LDH), abolished mitochondrial lactate consumption. Electrophoretic and fluorescence microscopic analyses helped localize LDH in the mitochondria. Taken together, this study implicates lactate as an important contributor to ATP metabolism in the brain, a finding that may significantly change our notion of how this important organ manipulates its energy budget

1.4 The Cerebral Tricarboxylic Acid Cycles

We review the operation of the cerebral tricarboxylic acid (TCA) cycles in the neuronal and glial compartments of the adult rat brain, with an emphasis on the mechanisms underlying intercellular oxidative coupling during glutamatergic neurotransmission. We begin with an update of the enzymatic properties, gene location, regulation, and regional distribution of the enzymes involved. Then, we describe the main methodologies used to investigate TCA cycle activity in vitro and in vivo such as autoradiography, positron emission tomography (PET), nuclear magnetic resonance (NMR) imaging or spectroscopy, and dual photon fluorescence microscopy. Previous interpretations conceived cerebral glucose metabolism during glutamatergic neurotransmission as a coupled process, involving exclusively anaerobic metabolism in the astrocytes and oxidative metabolism in the neurons. The glutamine cycle was proposed to be stoichiometrically coupled to astrocytic glucose uptake, glutamine synthesis being supported by astrocytic glycolysis only and glutamine being the main precursor of cerebral glutamate. Compelling evidences have accumulated since then, showing that astrocytes display significant oxidative capacity in vivo, more than 60% of the glutamine is produced from ATP synthesized by astroglial oxidative phosphorylation, and approximately 40% of cerebral glutamate is not derived from glutamine. Together, these findings suggest that the coupling mechanisms between astrocytic and neuronal oxidative and nonoxidative metabolisms are more complex than initially envisioned. In this review, we propose a novel mechanism based on the operation of intracellular redox switches and the transcellular coupling of the NAD(P)/NAD (P)H redox states between both cell types through lactate transfers. The redox switch/redox coupling hypothesis is compatible with the simultaneous operation of glycolytic and oxidative metabolisms in both neural cell types. Transcellular redox coupling through lactate transfers mimics the intracellular coupling existing between cytosolic NADH production and mitochondrial NADH oxidation, as seen from the redox shuttles exchanging reducing equivalents through the inner mitochondrial membrane of neural cells.This work was supported in part by grants SAF 2001‐224, SAF 2004‐03197, FISss C03/08, G03/155, C03/10 and PI051530 to S.C. JUSTESA IMAGEN S.A. provided the core support of LISMAR during this work. T.B. R was supported by a fellowship from Fundaçâo para a Ciência e Tecnologia, Portugal (SFRH/BD/5407/2001).Peer reviewe