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—The ketogenic diet is used as a treatment for different brain diseases. It involves replacement of dietary carbohydrates with fat, which leads to production of ketone bodies, predominantly β-hydroxybutyrate. The mechanism of the protective effect of the ketogenic diet remains poorly studied. In this work, the combined influence of glucose and β-hydroxybutyrate on the potentials of the synaptosomal plasma membrane and the mitochondrial membrane was investigated. It was found that the presence of ketone bodies in a glucose-containing incubation medium did not affect the synaptosomal plasma membrane potential, in contrast to what was previously observed for the neuronal plasma membrane. It was shown that incubation of synaptosomes in the medium containing only β-hydroxybutyrate resulted in mitochondrial depolarization (ΔΨ change). At the same time, the presence of ketone bodies in the glucose-containing incubation medium led to mitochondrial hyperpolarization. The observed depolarization was partially compensated by an increase in mitochondrial ΔpH, which was induced by mitochondrial depolarization as such, but not by the presence of β-hydroxybutyrate. Thus, ketone bodies can be utilized by synapses as energy substrates, but β-hydroxybutyrate is a less effective energy source than glucose.

In the brain, pH can be lowered in both healthy and disease states. Previously, we showed that moderate extracellular acidification (down to pHo 7.0), but not intracellular acidification, leads to mitochondrial depolarization in synaptosomes. This indicates that the plasma membranes of neuronal presynaptic endings have proton receptors that can induce mitochondrial dysfunction when activated. In the present paper we attempt to identify this hypothetical receptor. First, we have demonstrated that lowering pHo to 7.0 does not induce sodium influx as monitored by the fluorescent dye Sodium Green. This fact, in conjunction with the absence of calcium influx in the same conditions - demonstrated previously, excludes ion channels as possible receptors. However, we showed that acidification-induced mitochondrial depolarization is sensitive to thapsigargin - an inhibitor of calcium release from intracellular stores, U73122 - an inhibitor of phospholipase C, as well as Cu2+ and Zn2+, which can block the metabotropic proton receptor ovarian cancer G protein-coupled receptor 1 (OGR1). Furthermore, using fluorescent dye Fluo-3 we have demonstrated that moderate extracellular acidification induces a cytosolic calcium increase. Excess calcium was scavenged by mitochondria (monitored by fluorescent dye Rhod-2). Our results suggest that the metabotropic OGR1 is a hypothetical presynaptic receptor for low pH. Its activation leads to phospholipase C activation and calcium release from the endoplasmic reticulum followed by accumulation in mitochondria, which likely causes a decrease in mitochondrial membrane potential.

Ketoacidosis is a dangerous complication of diabetes mellitus in which plasma levels of ketone bodies can reach 20–25 mM. This condition is life-threatening. In contrast, a ketogenic diet, achieving plasma levels of ketone bodies of about 4–5 mM, can be used for treating different brain diseases. However, the factors leading to the conversion of the neuroprotective ketone bodies’ action to the neurotoxic action during ketoacidosis are still unknown. We investigated the influence of high concentration (25 mM) of the main ketone body, β-hydroxybutyrate (BHB), on intrasynaptosomal pH (pHi), synaptic vesicle cycle, plasma membrane, and mitochondrial potentials. Using the fluorescent dye BCECF-AM, it was shown that BHB at concentrations of 8 and 25 mM did not influence pHi in synaptosomes. By means of the fluorescent dye acridine orange, it was demonstrated that 25 mM of BHB had no effect on exocytosis but inhibited compensatory endocytosis by 5-fold. Increasing buffer capacity with 25 mM HEPES did not affect endocytosis. Glucose abolished BHB-induced endocytosis inhibition. Using the fluorescent dye DiSC3(5), it was shown that 25 mM of BHB induced a significant plasma membrane depolarization. This effect was not impacted by glucose. Using the fluorescent dye rhodamine-123, it was shown that BHB alone (25 mМ) did not alter the potential of intrasynaptosomal mitochondria. Importantly, the high concentration of BHB (25 mМ) causes the depolarization of the plasma membrane and stronger inhibition of endocytosis compared with the intermediate concentration (8 mM).

The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS., funded by the DFG (SFB779‐B14 and RTG2413 “SynAge” TP08), BMBF (IB‐049 “PrePLASTic”) and EU (MC‐ITN “ECMED”; LSA ESF ZS/2016/08/80645 “ABINEP”). C.I.S. is an editor for Journal of Neurochemistry

In the brain, pH can be lowered in both healthy and disease states. Previously, we showed that moderate extracellular acidification (down to pHo 7.0), but not intracellular acidification, leads to mitochondrial depolarization in synaptosomes. This indicates that the plasma membranes of neuronal presynaptic endings have proton receptors that can induce mitochondrial dysfunction when activated. In the present paper we attempt to identify this hypothetical receptor. First, we have demonstrated that lowering pHo to 7.0 does not induce sodium influx as monitored by the fluorescent dye Sodium Green. This fact, in conjunction with the absence of calcium influx in the same conditions - demonstrated previously, excludes ion channels as possible receptors. However, we showed that acidification-induced mitochondrial depolarization is sensitive to thapsigargin - an inhibitor of calcium release from intracellular stores, U73122 - an inhibitor of phospholipase C, as well as Cu2+ and Zn2+, which can block the metabotropic proton receptor ovarian cancer G protein-coupled receptor 1 (OGR1). Furthermore, using fluorescent dye Fluo-3 we have demonstrated that moderate extracellular acidification induces a cytosolic calcium increase. Excess calcium was scavenged by mitochondria (monitored by fluorescent dye Rhod-2). Our results suggest that the metabotropic OGR1 is a hypothetical presynaptic receptor for low pH. Its activation leads to phospholipase C activation and calcium release from the endoplasmic reticulum followed by accumulation in mitochondria, which likely causes a decrease in mitochondrial membrane potential.