Determination of mitochondrial metabolic phenotype through investigation of the intrinsic inhibition of succinate dehydrogenase

Determination of mitochondrial metabolic phenotype through investigation of the intrinsic inhibition of succinate dehydrogenase

Accepted Manuscript Determination of mitochondrial metabolic phenotype through investigation of the intrinsic inhibition of succinate dehydrogenase Al...

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Accepted Manuscript Determination of mitochondrial metabolic phenotype through investigation of the intrinsic inhibition of succinate dehydrogenase Alexander Panov, Zulfiya Orynbayeva PII:

S0003-2697(17)30395-0

DOI:

10.1016/j.ab.2017.10.010

Reference:

YABIO 12815

To appear in:

Analytical Biochemistry

Received Date: 19 December 2016 Revised Date:

11 October 2017

Accepted Date: 12 October 2017

Please cite this article as: A. Panov, Z. Orynbayeva, Determination of mitochondrial metabolic phenotype through investigation of the intrinsic inhibition of succinate dehydrogenase, Analytical Biochemistry (2017), doi: 10.1016/j.ab.2017.10.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Determination of mitochondrial metabolic phenotype through investigation of the

Alexander Panov1, Zulfiya Orynbayeva2

Institute of Molecular Biology and Biophysics, Russian Academy of Sciences, Siberian

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1

Branch, Novosibirsk 630117, Russia

Department of Surgery, Drexel University College of Medicine, Philadelphia, PA USA

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intrinsic inhibition of succinate dehydrogenase

Short Title. Determination of mitochondrial metabolic phenotype of SDH. Manuscript ABIO-16-912R1

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Institute of Molecular Biology & Biophysics Russian Federation, 630117

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Novosibirsk, Timakova St. 2/12

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Corresponding address: Alexander Panov

3647 N. Kimberly Drive, Atlanta, GA 30340 E-mail: [email protected] Tel. 404-210-7031 1

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Highlights:

mitochondrial respiration to the organ’s energy demands.

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Intrinsic inhibition of SDH is an important mechanism for attenuation of

The properties of intrinsic inhibition of SDH are organ and species specific.

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metabolically at the level of SDH.

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Energy production and generation of ROS by mitochondria are controlled

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Abstract Many diseases are accompanied by systemic or organ metabolic abnormalities. Therefore, investigation of the roles of mitochondrial dysfunction in the pathogenesis of major diseases

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requires a methodology that reflects the characteristics of mitochondrial metabolism particular for the organ of origin. We provide evidence that for brain and heart mitochondria the intrinsic inhibition of succinate dehydrogenase (SDH) is a key mechanism for attenuation of

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mitochondrial respiration and energy production in response to the organ’s energy needs. This mechanism also serves to minimize the production of reactive oxygen species when the organ is

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at rest. Changes in the organ’s workloads are accompanied by changes in metabolites that are used by mitochondria as substrates and for modification of energy production at the SDH level. Measurement of the respiratory activity of mitochondria with various substrates and substrate mixtures and use of bovine serum albumin as an SDH inhibitorwill be useful for evaluation of

Keywords:

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metabolic phenotype at the mitochondrial level.

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Mitochondria; brain; heart; succinate dehydrogenase; metabolism; ROS.

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1. Introduction Mitochondria are not only the major source of energy for most functions in cells and thus the whole body, but by necessity are also the center of many metabolic events. In the process

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called mitochondrial respiration mitochondria produce energy by “burning” hydrogen, derived from food. As an unavoidable complication of reduction of oxygen to water, during transport of electrons along the respiratory chain, some electrons become misplaced and produce oxygen

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radicals, which may exert deleterious effects on cellular structures. Of course, mitochondria are not the only source of reactive oxygen species, but they are probably the largest and most

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persistent source in tissues with a high respiratory rate. Also, mitochondria are the only place in the body where one type of “food” can be converted to others, and the intermediary metabolites can be used for different metabolic pathways and functions. Therefore, it is not surprising, that currently it is widely recognized that mitochondrial dysfunctions are important contributors to

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the pathogenesis of practically all major diseases.

To understand what roles mitochondrial dysfunctions play in various pathological processes, it is of paramount importance to study the basic mitochondrial function – respiration.

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Without understanding respiratory activities of mitochondria it is impossible to understand other

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processes in mitochondria and cells. Unfortunately, the current methodology of research on mitochondrial respiration in some respects has become outdated and even obsolete. In this article we present a new methodology based on three principal presumptions: 1. Mitochondria in vivo utilize not one, but a mixture of substrates; 2. Mitochondrial functions in each organ closely correlate with the organ’s functions and metabolism; 3. The intrinsic inhibition of succinate dehydrogenase (SDH) is not an in vitro artefact, but functions as a regulatory point for respiration and ROS production, depending on the energy demands of the organ. Both the

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hypotheses and the methodology we present in this paper have been developed based on our own experiments with normal and diseased animals. The effects we describe here are not only highly organ-specific but also depend on the metabolic phenotype of mitochondria which is unique for

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each species or strain of experimental animals. Below, we provide a brief description of the methods used to study the metabolic diversity of mitochondria from different organs and species of animals. The major purpose of this article is to resolve the contradictions related to the use of

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succinate as a substrate and the intrinsic inhibition of succinate dehydrogenase (SDH), which researchers encounter when working with isolated liver, heart and brain mitochondria.

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2. Methods

Previous basic mitochondrial research focused on methods of isolation and the study of mitochondrial functions such as respiratory activities, membrane potential measurements, Ca2+ sequestration, ROS production. These methods worked well and provided a large amount of

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fundamental information on how mitochondria function. During the last two or three decades, however, the goals of mitochondrial research have shifted to the roles of mitochondria in aging and pathogenesis of many diseases. Previous methods often stopped short of providing useful

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answers to the new questions. As it turned out, the problem was in the old methodology of how

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the mitochondria were incubated. A new understanding of mitochondrial metabolism and methods of incubation and analysis are needed which correlate with the organ’s functions and metabolism.

In this study we used the well-known classical methods of mitochondrial isolation [1, 2], determination of mitochondrial respiration [2, 3], measurements of membrane potential [2, 3] and reactive oxygen species (ROS) production [2, 3, 4]. In our experiments we used several different substrates and substrate mixtures in order to determine the optimal mitochondrial 5

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metabolic patterns (for example, the highest rates of O2 consumption and ATP production) for a specific organ or species of an animal. We used respiratory inhibitors only for analytical purposes.

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Animals - All animal use complied with the National Institutes of Health guidelines and was approved by the IACUC of Emory University (Atlanta, GA), and Carolinas Medical Center (Charlotte, NC). Male Sprague Dawley and Wistar rats from Taconic Farms Inc. (Germantown,

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NY 12526) were used for isolation of the liver, brain and heart mitochondria. In some experiments we used mice of FVB or C57Bl/6J strains. All animals were used at the age of 8

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weeks.

Isolation of mitochondria - Brain mitochondria were isolated from pooled forebrains of 2-3 rats using a method modified form that of Sims [1], as described in [2]. Rat heart and liver mitochondria were isolated as described in detail in [2]. The isolation medium consisted of: 75

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mM mannitol, 150 mM sucrose, 20 mM MOPS, pH 7.2, 1 mM EGTA. For experiments examining the effects of defatted bovine serum albumin (BSA), 0.1% BSA (Sigma A4503) was added to the isolation medium. The final suspensions of mitochondria were prepared using the

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incubation medium described below. With this medium, mitochondria in suspensions were much

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less prone to sedimentation and sticking together. Before taking a sample of mitochondria for any purpose, the suspension was thoroughly vortexed. Mitochondrial protein was determined with the Pierce Coomassie protein assay reagent kit. Simultaneous measurement of mitochondrial respiration and membrane potential Respiratory activities of the mitochondria were measured using a custom-made plastic minichamber of 560 µL volume equipped with a standard YSI (Yellow Spring Instrument Co., Inc.) oxygen minielectrode connected to a YSI Model 5300 Biological Oxygen Monitor, a 6

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custom-made tetraphenylphosphonium (TPP+)-sensitive minielectrode, and a KCl bridge to a Ag/AgCl reference electrode connected to a pH meter. The details of the methods and the chamber’s picture are published in [2]. All instruments were connected to the data acquisition

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system.

The incubation medium contained: 125 mM KCl, 10 mM MOPS, pH 7.2, 2 mM MgCl2, 2 mM KH2PO4, 10 mM NaCl, 1 mM EGTA, 0.7 mM CaCl2. At a Ca2+/EGTA ratio of 0.7, the free

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[Ca2+] is close to 1 µM as determined using Fura-2. Substrates were present in the medium from

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the beginning. Unlike liver mitochondria, which support endogenous respiration for 10-15 minutes, heart mitochondria support respiration on endogenous substrates for 2-3 minutes only, whereas brain and spinal cord mitochondria contain no endogenous substrates. When BM are added to the medium without substrate, there is no oxygen consumption. After addition of mitochondria to the suspension medium, we allow mitochondria to respire for two minutes in

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order to minimize the endogenous content of AMP, which is accumulated in mitochondria during the isolation and storage as the working suspension. ADP was added after 2 minutes of respiration in MS-4. We recommend the following substrate concentrations: 5 mM succinate

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(normally without rotenone), 5-10 mM glutamate, 2.5 mM pyruvate, 2 mM malate, and Lpalmitoyl-carnitine (in 50% ethanol) 25 µM. Oxidative phosphorylation (state 3) was initiated by

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addition of 150 µM ADP. The working concentration of CCCP for estimation of uncoupled respiration (state 3U) was determined by titration with CCCP (0.05 µM aliquots) until a maximum rate of oxygen consumption was obtained. Measurements of hydrogen peroxide generation - H2O2 was determined using Amplex red (Molecular Probes). The details of the method are described in [2, 3]. Additions of resorufin were used for calibration of the fluorescence scale. Fluorimetric measurements were made using 7

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a

highly

sensitive

fluorometer

from

C&L

Company,

Middletown,

Pennsylvania

(www.fluorescence.com). Data acquisition - Data acquisition was performed using hardware and software from C&L

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Company.

Chemicals - Chemicals were of the highest purity available. All solutions were made using glass bidistilled water. Since filter sterilization of media usually resulted in extraction into

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cold room and used it for no longer than 7-10 days.

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solution of high level of peroxides [2], we stored the freshly prepared non-sterilized media in the

Statistics - Comparisons between two groups were made by unpaired t-test and comparisons between more than two groups were made by ANOVAs followed by post hoc tests. 3. A few words about the current basic theoretical concepts. Since we will discuss

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mitochondrial physiology not only as a source of ATP, but in a much wider sense, keeping in mind all metabolic events and transformations of different forms of energy in cells and organisms, we have to rely on thermodynamic laws of irreversible processes and the related

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concepts. This is important because practically all metabolic pathways are coupled to each other and occur simultaneously. This is possible only because most important energy-dependent

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reactions in metabolic pathways are compartmentalized and have one or more points of irreversibility. For example, phosphorylation of ADP to ATP occurs in the matrix of mitochondria, whereas most ATP-consuming reactions occur in the cytoplasm. We also have to keep in mind that in mitochondria most enzymes are organized in dynamic complexes, often very stable, when the intermediary metabolites are passed from one enzyme to another without being actually released in a free form [5-7]. Therefore, it is not productive to think in terms of concentrations of substrates in vivo, instead, we have to consider fluxes and rates [8]. Therefore, 8

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we also have to remember that in the in vitro system, metabolism of the externally added substrates may have different kinetic and thermodynamic properties as compared to the situation in vivo, when metabolites are transferred directly not only from one enzyme to another, but also

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from one metabolic pathway to another.

Because mitochondria are open systems, and every organ may have various metabolic patterns, mitochondria from different organs may have diverse mixtures of metabolites entering

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and exiting the citric acid cycle (TCA), and electrons may enter the respiratory chain at different levels and from different metabolic pathways (Fig. 1). In addition, acetyl-CoA - the starting

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metabolite of the TCA cycle, much more commonly originates from fatty acids rather than from pyruvate [2, 9]. Thus it would make sense in experiments with isolated mitochondria to use mixtures of substrates that would correspond to the organ’s metabolic patterns. For example, for the isolated brain mitochondria the physiological substrate mixture can be pyruvate + glutamate,

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whereas for the heart mitochondria it can be palmitoyl-carnitine + glutamate or pyruvate. In both examples we observed much higher rates of oxidative phosphorylation than with traditional substrates glutamate + malate or pyruvate + malate [2].

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4. Succinate is a substrate for mitochondrial respiration, but SDH is not the

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respiratory complex. In mitochondria oxidizing succinate + rotenone, energization is so efficient that the enzyme SDH was named “the respiratory complex II”, which provides electrons to the respiratory chain at the level of complex III. Thus, according to the old paradigm, with succinate as a substrate, there were only two active “coupling sites” (later named proton pumps), instead of three “coupling sites” with the “classical” NAD-dependent complex I substrates glutamate or pyruvate.

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However, if we look at the respiratory chain from a functional point of view, we see practically no difference between SDH and the acyl-CoA dehydrogenase complex, which is comprised of three enzymes: acyl-CoA dehydrogenase, electron-transport flavoprotein (ETFP),

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and ETFP dehydrogenase. Together, this complex forms the trans-double bond between C2 and C3 atoms during β-oxidation of fatty acids. The third similar enzyme is the mitochondrial isoform of sn-glycerol-3-phosphate dehydrogenase (mGPDH). All these enzymes contain FAD

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as a cofactor and reduce the membrane pool of ubiquinone. The most important distinction between this group of FAD-dependent enzymes and the true respiratory chain complexes I, III

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and IV, is that the FAD-enzymes do not directly participate in generation of the transmembrane electrochemical potential (∆p = ∆Ψ -59 ∆pH).

Although SDH and other FAD dehydrogenases under discussion do not participate directly in the generation of ∆p, it is very important that by transferring electrons to the respiratory chain

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via the membrane’s pool of reduced coenzyme Q, this group of enzymes supplies hydrogen for respiration and thus introduces irreversibility to their corresponding metabolic pathways. This is a very important function of these FAD enzymes because it has a great impact on the reverse

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electron transport and the associated production of ROS [4, 10, 11].

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Figure 1 shows the position of succinate dehydrogenase (SDH) within the sequence of enzymes of the tricarboxylic acids (TCA) cycle, which is organized into multienzyme structures [12, 13]. Succinate is an intermediate during “normal” work of the TCA cycle, when the starting metabolite is acetyl-CoA derived either by decarboxylation of pyruvate or, much more commonly, during β-oxidation of fatty acids. Therefore it was generally considered that formation and further metabolic transformations of succinate are stoichiometric to the general activity of the TCA cycle.

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Figure 1 also shows that the TCA cycle is connected to a number of anaplerotic and catabolic pathways, which remove from, or supply metabolites into the cycle. In some organs such as the brain and heart, during oxidation of glutamate or pyruvate, the TCA cycle may

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function as two distinct coupled subcycles (Fig. 2) [14]. This occurs due to interactions of the TCA cycle with aspartate aminotransferase (AST), alanine aminotransferase (ALT) and the malate/aspartate shuttle (MAS). Yudkoff et al. [14] have shown that metabolites in the subcycle

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A may turn over five times faster than in the subcycle B (Fig. 2). The reactions catalyzed by AST and ALT consume oxaloacetate (OAA) and thus “short-circuit” the TCA cycle. This results in a

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several-fold higher rate of α-ketoglutarate production, which is then converted to succinate, fumarate and malate, as compared to the turnover of metabolites in the subcycle B. According to Balazs [15], in brain mitochondria oxidizing a mixture of glutamate + pyruvate + malate the level of α-ketoglutarate increases 30-fold! Panov et al. [3] have shown that with isolated brain

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and spinal cord mitochondria, up to 50% of added glutamate or pyruvate are oxidized through the transamination pathway producing succinate that stimulates reverse electron transport. Simultaneous oxidation by neuronal mitochondria of pyruvate, provided by astroglia, and the

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neuromediator glutamate is a physiologically normal situation during activation of synapses [3, 9]. Catabolism of the inhibitory neuromediator γ-aminobutyric acid (GABA), which is

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transported from synaptic clefts to synaptosomal mitochondria, may serve as an additional source of succinate for some neurons in brain and spinal cord [16]. Thus, mitochondria in different organs and even in different cells of the same organ, such as brain, may be subjected to different fluxes of succinate. This may have important consequences to organs such as brain and heart, which are particularly susceptible to oxidative

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stress because currently the succinate-supported reverse electron transport is considered as one of the most important sources of oxidative stress, [3, 9, 17-19]. 5. Properties of succinate dehydrogenase (SDH). Because of tight interactions of SDH

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with the endogenously formed oxaloacetate (OAA), it is difficult to determine the catalytic properties of the isolated SDH. The major difficulty is that during the isolation procedure, SDH loses activity due to the almost irreversible binding of OAA [20]. Vinogradov et al. studied

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properties of the isolated SDH and showed that OAA binds to the SH group of cysteine in the enzyme’s active center [21]. The binding has two steps: first, OAA reacts with SH group of SDH

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and competitively inhibits the enzyme’s activity toward succinate; then, the double bond of OAA (enol form) reacts covalently with cysteine, and the inhibition becomes almost irreversible. Experiments with the isolated enzyme have shown that during the inactivation step, binding of OAA is accompanied by a large decrease of the FAD’s midpoint redox potential, which renders

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the oxidation of succinate impossible. Removal of OAA increases the redox potential of FAD and thus enables the binding and oxidation of succinate [22]. Hirst et al. [23] showed that it is possible to restore FAD in the active center of the isolated SDH, which was absorbed on a

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pyrolytic graphite edge electrode rode. Using this method, Hirst et al. were able to make SDH function either in the direction of succinate oxidation or fumarate reduction [23]. The catalytic

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activity of the enzyme dropped sharply as the potential became more negative. This is an intrinsic property of SDH that is associated with the two-electron/two-proton reduction of FAD. The redox state of FAD strongly affects binding and release of the competitive inhibitor/regulator oxaloacetate. Oxidation of FAD causes tight binding of OAA, while reduction of FAD weakens binding of OAA [23]. These data indicate that, in principle, the electron transport from FAD to Coenzyme Q can be reversed [23], but this usually does not occur in

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living cells because of continuous oxidation of the membrane’s pool of CoQH2 by the respiratory chain of mitochondria, making the associated metabolic pathways irreversible. Unlike experiments with isolated SDH or submitochondrial particles [8, 9], in experiments

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with isolated intact mitochondria, even the strongest intrinsic inhibition of the enzyme by OAA is very quickly eliminated in the presence of substrates that remove OAA metabolically in the transaminase reactions [3, 24]. This suggests that in vivo OAA does not bind covalently to the

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SH group in the active center. Another important feature of the intrinsic SDH inhibition by OAA in “live” mitochondria is that the strength of the OAA binding varies strongly among

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mitochondria isolated from various organs and species [24]. Addition of defatted BSA to the isolation buffer removes the intrinsic inhibition, and in some animals, brain mitochondria, isolated with BSA, the high rates of succinate oxidation are restored in MS-3 and MS-4. However, after storage of BM on ice for 1-2 hrs., the inhibition of SDH may return (A. Panov,

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unpublished data).

In experiments in vitro, it has been established that malate, when added to mitochondria at 1-2 mM concentration, is a more efficient inhibitor of succinate oxidation than is added OAA (1-

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2 mM). At high concentrations of succinate (5 mM) there is a slow competitive inhibition of

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SDH by OAA. Malate inhibits SDH non-competitively, but the effect is highly dependent on the origin of the mitochondria. The inhibition is particularly evident with brain mitochondria. According to Vinogradov et al. [21], oxidation of SDH results in a 10-fold increase in affinity toward OAA. This is the reason why, with succinate alone, addition of ADP or uncoupler to mitochondria causes inhibition of respiration. The differences between OAA and malate as inhibitors of SDH observed in vitro, to a large extent may be explained by variations in affinities of these metabolites to the enzyme. OAA 13

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binds to SDH competitively with succinate, whereas malate is a substrate for SDH with a similar affinity to the enzyme as succinate [25, 26]. Purified succinate-ubiquinone reductase catalyzes oxidation of both L- and D-malate with Km of about 2 mM and initial Vmax of 50 and 100

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nmol/min/mg protein for L- and D-stereoisomers, respectively (25o C, pH 7.0). Oxidation of malate by isolated SDH in the presence of the OAA trapping system occurs at an indefinitely constant rate when enolOAA, which is an immediate product of the reaction, is rapidly converted

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into the keto isomer, which is a substrate for aspartate aminotransferase [26]. Oxidation of malate by SDH leads to formation of enolOAA directly on the active center and thus causes

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inhibition of the enzyme. The metabolic accumulation of OAA and malate in resting mitochondria may be responsible for the “intrinsic inhibition” of SDH. As we have mentioned above, addition of defatted bovine serum albumin to the isolation or incubation media can remove OAA from brain mitochondria and thus release the intrinsic inhibition of SDH. The

organ-specific [24].

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situation is different with the heart mitochondria. This is because the effects of BSA are also

6. Intrinsic inhibition of SDH in brain mitochondria and the effects of BSA. From the

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very beginning of our work with brain mitochondria, it was clear that the presence of 0.1% defatted BSA in the isolation medium might somehow affect the intrinsic properties of

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mitochondria due to the well-known ability of BSA to bind a wide spectrum of ions, various hydrophobic, compounds, hormones and peptides [27, 28]. There were other reasons to study the effects of defatted BSA on mitochondrial functions and succinate oxidation in the absence of rotenone more closely. BSA indeed directly affects mitochondrial functions, and the effects are dependent on the type of organ and the metabolic phenotype of an animal [24]. Needless to say, there is no rotenone present in the tissues of animals, so we had to understand what is happening

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to mitochondria oxidizing succinate in the absence of rotenone. In addition, rotenone prevents measurements of ROS production associated with the succinate-driven reverse electron transport. Figure 3A illustrates the fact that brain mitochondria from FVB mice (MBM) isolated in

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the presence of 0.1% BSA (BSA-MBM) show high membrane potential (lower line) and relatively high rates of succinate (no rotenone) oxidation in MS-4 and in MS-3 (upper line). In the MBM isolated without BSA (nonBSA-MBM) oxidation of succinate in MS-4 is so slow, that

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membrane potential could not be generated at all (Fig. 3B). Addition of glutamate instantly activates respiration and membrane potential reaches its maximum in just about 10 seconds (Fig.

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3B). This indicates that the lack of ability of the nonBSA-MBM to oxidize succinate and generate membrane potential is not caused by any damages to the mitochondria, except inhibition of SDH by oxaloacetate. It must be mentioned, that unlike mitochondria from other organs, isolated mitochondria from rodent brain and spinal cord possess no endogenous

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substrates [2].

A thorough description of the inhibition of SDH by endogenous OAA and the effects of BSA are presented in [24]. Here we can mention that the degree of OAA-dependent intrinsic

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inhibition of SDH varies strongly among species, and organs. FVB mice and Sprague Dawley

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rats are among the species that exhibit very strong intrinsic inhibition of SDH in brain mitochondria. However, the metabolic pattern of mitochondria, even for the same strain, which we have ordered from the same company for a number of years, changed with time, and this may have large experimental consequences, as described in our paper [29]. Evidently, the degree of intrinsic inhibition is determined by the affinity of SDH toward OAA, which is subject to genetic modifications. With brain mitochondria, BSA is capable of eliminating inhibition by removing

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OAA from the enzyme during the isolation procedure. With rat heart mitochondria, however, the effects of BSA on intrinsic inhibition of SDH are different. 7. Intrinsic inhibition of SDH and the effects of BSA in heart mitochondria. Data

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presented in Figures 3 and 4 illustrate variations of the intrinsic inhibition of SDH and responses to BSA of the mouse brain (Fig. 3) and rat heart (Fig. 4) mitochondria (Sprague Dawley). Unlike the intrinsic inhibition of SDH in brain mitochondria, which is sensitive to BSA, rat heart

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mitochondria (RHM) are insensitive to the presence of BSA in the isolation medium. Figure 4A shows that in RHM, oxidizing succinate, both the rate of O2 consumption in MS-4 and

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membrane potential (∆Ψ) are high, but addition of either ADP or uncoupler (CCCP) causes a strong decline in respiration and ∆Ψ, which is rapidly eliminated by addition of glutamate + malate. This experimental features is observed regardless of whether RHM are isolated with or without BSA. Figure 4B shows that oxidation of succinate in the presence of glutamate + malate

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results in high rates of respiration both in MS-3 and MS-3U.

8. Effects of malate on ROS production by brain and heart mitochondria oxidizing various substrates and substrate mixtures.

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We have mentioned above that malate, a natural metabolite of the TCA cycle, can be

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oxidized by SDH with formation of oxaloacetate directly on the enzyme’s active center [26]. This makes malate a potentially important intracellular regulator of the mitochondrial resting respiration and ROS production by the succinate-dependent reverse electron transport. In figures 3 and 4 we show that addition of either glutamate alone (Fig. 3) or together with malate (Fig. 4) almost instantly releases the intrinsic inhibition of SDH. Further investigations have shown that the release of SDH inhibition does not depend on the presence of malate becauseit requires only the presence of a substrate for transaminase reactions (glutamate or pyruvate). However, malate 16

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greatly affects the rates of respiration in MS-4 after activation of SDH and the associated production of ROS. Figure 5 shows the effects of malate on ROS production by rat brain mitochondria (RBM)

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(Fig. 5A) and RHM (Fig. 5B) oxidizing various substrates and substrate mixtures. Both RBM and RHM were isolated without BSA in the isolation medium; the rates of ROS production with pyruvate + malate are taken as 100%.

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With glutamate + malate, RBM produce less ROS than with pyruvate + malate (Fig. 5A).

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Earlier, we showed that a mixture of pyruvate + glutamate + malate resembles the metabolic situation for nonsynaptic mitochondria in the activated brain [3]. Fig. 5A shows that with this substrate mixture the rate of ROS production increases two-fold. With succinate alone, RBM produces ROS close to the level observed with pyruvate. When succinate is oxidized in the presence of either pyruvate or glutamate, but without malate, the rates of ROS production

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increase 5 times. However, when malate is also present, there is significant inhibition of ROS production, including with succinate alone (Fig, 5A). With RHM, the rates of ROS production with pyruvate and glutamate are similar and do

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not increase when oxidized simultaneously (Fig. 5B). This proves that stimulation of respiration

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in all metabolic states and ROS production in the presence of pyruvate + glutamate is a specific metabolic feature of the brain mitochondria only. Unlike RBM, ROS production by RHM, oxidizing succinate alone, is 7.7-fold higher than that observed with with pyruvate, and increases 11-fold, when RHM oxidize the substrate mixture succinate + glutamate + pyruvate. When malate is added to this substrate mixture, ROS production increases only 5-fold, a 64% inhibition as compared with that observed with succinate + glutamate + pyruvate.

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The results presented in Fig. 5 clearly show that malate can significantly affect succinatedependent ROS production. However, the effects of malate vary greatly among different species. We have presented an example of how relatively slight changes in the metabolism of succinate

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and malate in brain mitochondria affect the sensitivity of transgenic animals to the human mutated gene SOD1 [29]. Recently, interesting data have been published on the in vivo effects of dietary administered malate [30, 31]. Wu et al. [30] have shown that dietary L-malate reducesthe

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accumulation of ROS and significantly decreases the level of lipid peroxidation in the liver and heart of aged rats. Kolosova et al. [31] studied the effects of dietary malate on the brain structure

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of the senescence-accelerated OXYS rats. These animals are well-known for early development of aged phenotype, including the stereotyped behavior specific for aging animals [32]. Kolosova et al. [31], using magnetic resonance imaging (MRI), confirmed the presence of neurodegenerative brain changes in young OXYS rats. Malate had a pronounced protective

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effect in the prevention of early brain tissue degradation and fully prevented the appearance of new demyelination foci caused by rotenone intoxication. Thus, the animal studies demonstrated the use of malate as a prospective neuroprotector [30, 31]. These experiments are in full

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agreement with our proposition that intrinsic inhibition of SDH plays important roles in diminishing or protection from damages caused by oxidative stress.

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9. Discussion.

The physiological roles of intrinsic SDH inhibition and the metabolic phenotype of the mitochondria under investigation can be understood only if a researcher has information about several, if not all, major functions of mitochondria. The respiratory activities in different metabolic states with various substrates and substrate mixtures [9], as well as the dynamics of

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changes of membrane potential and ROS production with each substrate mixture, are important mitochondrial functions [3]. The experiments, presented in figures 3 and 4, clearly emphasize the fact that inhibition of

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succinate oxidation is not associated with any dysfunctions in the respiratory chain or other damage to mitochondria. Because the intrinsic SDH inhibition is almost instantly eliminated metabolically by glutamate or pyruvate, we can suggest that inhibition of SDH may have

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important physiological and regulatory functions. We suggest that one of the physiological and pathophysiological significances of the intrinsic SDH inhibition may be the prevention of

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excessive production of ROS in the resting brain and heart. Inhibition of SDH prevents or diminishes the reverse electron transport, which is “automatically” removed metabolically as soon as neurons or heart become activated and receive additional portions of substrates that metabolically consume OAA. Data in support of this hypothesis have been published in a

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number of papers [2-4, 9, 29].

One interesting conclusions, which we made from a number of experiments, is that the rates of oxygen consumption during resting respiration (MS-4) correlate with the rates of ROS

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production [3, 9]. Increased oxygen consumption in MS-4 with some substrate mixtures could be

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interpreted as an indication of uncoupling of respiration. For example, mixtures of succinate with either glutamate (see Fig. 4A, 4B), or palmitoyl-carnitine may significantly increase both MS-4 respiration and ROS production [9]. Reverse electron transport is an energy-dependent function and during the formation of superoxide radicals, O2 serves as a sink for electrons, and when formed the O2•− anion leaves the membrane and thus promotes production of new radicals. However, the proposition that succinate-induced ROS production during resting respiration of isolated mitochondria may also occur in vivo met a serious objection [33]. Starkov & Fiskum 19

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[33] correctly noted that since the reverse electron transport is an energy-dependent function, and that in vivo mitochondria are always “busy” with generating ATP, maintaining membrane potential, transport of anions and cations, the steady state membrane potential will be

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significantly diminished and thus prevent the reverse electron transport and the associated ROS production. This objection is absolutely correct for the in vivo situation in most organs, except for the brain, heart and skeletal muscles. Heart and, particularly, skeletal muscles have a very

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high workload range [34]. It is well known that the heart and brain are the most sensitive organs in regard to aging and pathologies caused by oxidative stress.

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Abeles [35] stressed that in brain a vast majority of mitochondria are located at synaptic junctions, and their only function is to produce large amounts of ATP in order to restore, in a fraction of a second, the balance of ionic concentrations across the membranes of activated synapses. When the neurons are at rest, there is no other energy-consuming functions for

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mitochondria located around synapses, and they may become hyperpolarized and stimulate the reverse electron transport and the associated production of ROS [3]. Catabolism of GABA and transamination of pyruvate and glutamate, which are normal substrates for neuronal

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mitochondria, may significantly increase the flux of succinate in synaptic mitochondria and ROS production in resting neurons (Fig. 5A). Thus, the intrinsic inhibition of SDH may serve as an

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automatic regulator of respiration and ROS generation: at rest, the neuronal mitochondria have high ∆p, but low reverse electron transport and low ROS production. However, upon neuronal activation, the increased flux of pyruvate (formed from lactate provided by astrocytes) and the neuromediator glutamate from the synaptic cleft, metabolically remove OAA and thus activate oxidative phosphorylation [3, 15].

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In regards to heart mitochondria, it is well known that with age many people are prone to development of the so called “Metabolic syndrome” and increased risk of developing cardiovascular pathologies [36]. From the point of view of mitochondrial metabolism, the Metabolic

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syndrome is associated with increased utilization of fatty acids as substrates for mitochondrial respiration. Our data have shown that both in the heart and brain mitochondria oxidation of substrate mixtures, which included palmitoyl-carnitine (25 µM), is accompanied by a significant

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increase in respiration in MS-4 and the production of ROS [9, 37]. One of the major features of energy metabolism in the heart is that the heart has a very large workload range. We suggest, that

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in the sedentary heart mitochondria may be hyperpolarized and produce more ROS. For this reason, intrinsic inhibition of SDH in the heart mitochondria may also serve as a regulatory mechanism of the respiratory activity and ROS production and participate in the anaplerotic and cataplerotic functions of the TCA cycle [38]. This hypothesis, however, requires further

10. Conclusions.

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experimental work.

The data presented in this paper suggest that intrinsic inhibition of SDH by OAA is an

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important mechanism of regulation of the overall respiratory activity of mitochondria in neuronal

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and heart mitochondria. This mechanism allows the adjustment of respiration and thus production of energy to the current energy needs of the organ and minimizes production of ROS when the organ is at rest. For many neurodegenerative diseases, for example Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis, the so called sporadic cases represent more than 90% of all cases [39]. Researchers, however, often study the relatively few cases which have some genetic background. In this paper we present a new methodology, which allows us to study regulation of mitochondrial activity and determine the mitochondrial

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metabolic phenotype using SDH as a target enzyme. This approach to evaluating mitochondrial functions opens a new pathway to study pathologies, which have no evident association with mutations of nuclear DNA or mitochondrial DNA.

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The essence of the methodology presented in this paper is based on the investigation of mitochondria as they are, that is, mitochondria that are isolated without any protective addition of BSA or any other compound. Measurements of respiratory activities are conducted with

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various metabolites and their mixtures which reflect the metabolic activities of the organ under investigation. BSA and inhibitors are used for the sake of comparison and analysis. The

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experimental details of this methodology for investigation of brain mitochondria from normal

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and diseased animals are presented elsewhere [2, 3, 4, 19].

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11. Acknowledgement. The authors express their gratitude to Mrs. Nancy Fox Ciliax for proofreading the text of

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the manuscript.

12. References

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Abbreviations: ADP – adenosine diphosphate, ATP – adenosine triphosphate, ALT – alanine aminotransferase, AST – aspartate aminotransferase, BSA – bovine serum albumin; BSA-BM or BSA-HM – brain or heart mitochondria isolated in the presence of 0.1% defatted

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BSA, CCCP - carbonyl cyanide m-chlorophenyl hydrazine, CoQH2 – reduced coenzyme Q, EGTA - ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ETFP - electrontransport flavoprotein,

FAD - flavin adenine dinucleotide, GABA - γ-aminobutyric acid,

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mGPDH - sn-glycerol-3-phosphate dehydrogenase, IMM – inner mitochondrial membrane, MS – mitochondrial respiratory metabolic states: MS-4 resting respiration, mitochondria have - active oxidative phosphorylation of added ADP, MS-3U –

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maximal energization, MS-3

uncoupled respiration; OAA - oxaloacetate, OXYS – rats with senescence-accelerated features, derived genetically from Wistar rats, ROS – reactive oxygen species, SDH – succinate dehydrogenase, SOD1 -superoxide dismutase outside mitochondria and SOD2 – mitochondria

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superoxide dismutase, TCA cycle – tricarboxylic acid cycle (the Krebs cycle).

Legends to the figures.

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Figure 1. Position of succinate dehydrogenase within the chain of reactions of the

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tricarboxylic cycle.

The TCA cycle is presented with the major anaplerotic and cataplerotic reactions illustrating that the TCA cycle is an open system and serves as a source of metabolites for anaplerotic (citrate, OAA, α-ketoglutarate) reactions, or serves as a sink for the final products of the catabolic pathways (amino acids). In every organ or tissue the TCA cycle is adapted to the metabolic needs of the host organ. The figure was modified from that published in an open access paper [38], we added only an indication that fatty acids also serve as an important source 29

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for acetyl-CoA. In many organs, for example, in the liver, heart and kidney, fatty acids are the main source of acetyl-CoA. In the brain, at least 20% of the total energy production is supported

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by oxidation of fatty acids [reviewed in 9].

Figure 2. Functioning of the TCA cycle as two coupled minicycles.

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In some tissues, for example in the heart and brain, the TCA cycle functions as two coupled minicycles (A and B). When mitochondria oxidize a mixture of substrates containing pyruvate

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and/or glutamate, the turnover of metabolites in cycle A may be several times faster, than in cycle B. Transamination of pyruvate or glutamate consumes OAA

with production of α-

ketoglutarate, and thus “short-circuits” the TCA cycle, and removes the endogenous inhibition of SDH. The image of a lock indicates a quasi-irreversible inhibition of SDH by malonate; the

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dashed lines indicate that OAA and malate are also inhibitors of SDH.

Figure 3. Oxygen consumption and membrane potential exhibited bу mouse (FVB)

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brain mitochondria isolated with and without BSA. Line 1 (black) – O2 consumption; Line 2 (red) – membrane potential. А. Forebrain

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mitochondria were isolated in the presence of 0.1% BSA; В. Brain mitochondria were isolated without BSA. Incubation conditions: 125 mM KCl, 10 mM MOPS, pH 7.2, 2 mM MgCl2, 2 mM KH2PO4, 10 mM NaCl, 1 mM EGTA, and 0.7 mM CaCl2, succinate 5 mM. Additions: ADP 150 µМ, СССР 0.5 µМ, glutamate 10 мМ (no malate). The release of SDH inhibition by glutamate does not depend on the presence of malate.

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Figure 4. Oxidation of succinate and glutamate by isolated heart mitochondria obtained from Sprague Dawley rats. Incubation conditions and additions are as in Fig. 3. Fig. 4A - rat heart mitochondria

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oxidizing succinate; Fig. 4B – rat heart mitochondria oxidizing succinate + glutamate + malate. The results presented in figures 4A and 4B are very similar regardless of the presence of BSA in the isolation medium. Respiratory rates in Fig. 4A and 4B rates are presented in nmol O2/min/mg

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protein.

Figure 5. Effects of malate on ROS production by rat brain and heart mitochondria oxidizing various substrates and substrate mixtures.

Incubation conditions are as in Figure 3, substrate concentrations are as described in the

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Methods. The data are the average of three different isolations (M±St. error). ROS production was measured with the Amplex red method as described in [2]. ROS production with pyruvate + malate was taken as 100%. A - Rat brain mitochondria. B - Rat heart mitochondria. Statistics:

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*** - p<0.001; NS – non significant. All differences are comparisons with pyruvate + malate.

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Abbreviations: P – pyruvate, G – glutamate, M – malate, S – succinate, Mln – malonate 5 mM

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Figures.

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Figure 1.

Figure 2.

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Figure 3.

Figure 4.

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Figure 5.

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