Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ_m) and pH gradient (ΔpH_m) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH_m, whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH_m in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (∼7.6) and ΔpH_m (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH_m decreased during cytosolic Ca²⁺ elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca²⁺-ATPases and transmitted to mitochondria by P_i/H⁺ symport and K⁺/H⁺ exchange, whereas the decrease in ΔpH_m reflected the low H⁺-buffering power of mitochondria (∼5 mm, pH 7.8) compared with the cytosol (∼20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca²⁺ and pH, mitochondria alkalinized and ΔpH_m increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca²⁺ caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH_m are regulated by opposing Ca²⁺-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.

Medium‐chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium‐chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell‐derived human astrocytes and neurons. Using single‐cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP‐activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17‐fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.—Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium‐chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte‐neuron lactate and ketone body shuttle systems. FASEB J. 30, 1913–1926 (2016). www.fasebj.org

During cell activation, mitochondria play an important role in Ca²⁺ homoeostasis due to the presence of a fast and specific Ca²⁺ channel in its inner membrane, the mitochondrial Ca²⁺ uniporter. This channel allows mitochondria to buffer local cytosolic [Ca²⁺] changes and controls the intramitochondrial Ca²⁺ levels, thus modulating a variety of phenomena from respiratory rate to apoptosis. We have described recently that SB202190, an inhibitor of p38 MAPK (mitogen-activated protein kinase), strongly activated the uniporter. We show in the present study that a series of natural plant flavonoids, widely distributed in foods, produced also a strong stimulation of the mitochondrial Ca²⁺ uniporter. This effect was of the same magnitude as that induced by SB202190 (an approx. 20-fold increase in the mitochondrial Ca²⁺ uptake rate), developed without measurable delay and was rapidly reversible. In intact cells, the mitochondrial Ca²⁺ peak induced by histamine was also largely increased by the flavonoids. Stimulation of the uniporter by either flavonoids or SB202190 did not require ATP, suggesting a direct effect on the uniporter or an associated protein which is not mediated by protein phosphorylation. The most active compound, kaempferol, increased the rate of mitochondrial Ca²⁺ uptake by 85±15% (mean±S.E.M., n=4) and the histamine-induced mitochondrial Ca²⁺ peak by 139±19% (mean±S.E.M., n=5) at a concentration of 1 μM. Given that flavonoids can reach this concentration range in plasma after ingestion of flavonoid-rich food, these compounds could be modulating the uniporter under physiological conditions.

Mitochondria capture and subsequently release Ca(2+) ions, thereby sensing and shaping cellular Ca(2+) signals. The Ca(2+) uniporter MCU mediates Ca(2+) uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca(2+) against Na(+) or H(+), respectively. Here we study the role of these ion exchangers in mitochondrial Ca(2+) extrusion and in Ca(2+)-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca(2+) efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca(2+) ([Ca(2+)]mt) elevations. NCLX overexpression enhanced the rates of Ca(2+) efflux, whereas increasing LETM1 levels had no impact on Ca(2+) extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca(2+)]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The [Ca(2+)]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na(+)/Ca(2+) exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca(2+) extrusion from mitochondria. By controlling the duration of matrix Ca(2+) elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca(2+) signals into redox changes.