387 ISSN 0006-3509, Biophysics, 2019, Vol. 64, No. 3, pp. 387–392. © Pleiades Publishing, Inc., 2019. Russian Text © The Author(s), 2019, published in Biofizika, 2019, Vol. 64, No. 3, pp. 500–506. The Combined Effect of Glucose and β-Hydroxybutyrate on the Membrane Potential of Synaptosomal Mitochondria T. G. Dubouskaya a , S. V. Hrynevich a , and S. V. Fedorovich a, * a Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Minsk, 220072 Belarus *e-mail: fedorovich@ibp.org.by Received June 26, 2017; revised October 1, 2018; accepted March 18, 2019 Abstract—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-hydroxybutyr- ate. 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 mem- brane 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. Keywords: synaptosomes, ketogenic diet, ketone bodies, mitochondria, membrane potential, synapse DOI: 10.1134/S0006350919030060 A ketogenic diet is used in the treatment of differ- ent diseases affecting the central nervous system, first of all, epilepsy and Alzheimer’s disease [1–3]. Its basic principle involves replacement of dietary carbohy- drates with fats. In the liver fats are transformed into ketone bodies, primarily β-hydroxybutyrate (BHB) and acetoacetate, which may further be utilized by brain cells as energy substrates. The main ketone body component is BHB. At the same time, blood glucose levels decrease [1–3]. The mechanism underlying the protective effect of the ketogenic diet still remains unclear. A number of potential targets of ketone bodies have been discussed, such as histone deacetylase (epigenetic action), plasma membrane potential, and potassium channels [1–3]. In our recent study, we found that glucose replacement with BHB in the incubation medium led to inhibition of endocytosis in the presynaptic nerve endings, which may underlie the anticonvulsant effect of the ketogenic diet [4]. We did not observe any influ- ence of ketone bodies on the plasma membrane poten- tial [4]. On the other hand, in the presence of glucose, BHB induced hyperpolarization in neurons [5]. Therefore, further experiments are required to investi- gate combined effects of glucose and BHB. Previously, it was shown that presynaptic nerve endings can utilize BHB as an energy substrate and oxidize it to CO 2 [6]. However, it remains uncertain whether ketone bodies suffice to maintain the poten- tials of both the cell membrane and the synaptic mito- chondria. We found that under hypoglycemic condi- tions, the mitochondrial potential changed much more strongly than the cell membrane potential. Moreover, a decrease in ΔΨ was compensated by an increase in ΔpH [7]. It is unknown whether the same effect occurs if synaptosomes utilize ketone bodies as an energy substrate. In this work, we investigated the combined effect of glucose and BHB on the plasma membrane potential in isolated presynaptic nerve endings and the mem- brane potential of synaptosomal mitochondria. The study was performed using synaptosomes, that is, isolated presynaptic nerve endings that retain the principal properties of intact terminals [8]. They are capable of exo- and endocytosis, maintain the plasma membrane potential, and contain active mitochondria [7–9]. The mitochondrial membrane potential is a cumulative biophysical parameter that adequately reflects their functional activity. Abbreviations: BHB, β-hydroxybutyrate, CCCP, carbonyl cya- nide 4-(trifluoromethoxy)phenylhydrazone; DiSC3(5), 3,3’- dipropylthiadicarbocyanine. CELL BIOPHYSICS