Succinate as a mobilization cue

Eugene I. Maevsky, Anna A. Vasilyeva, Mikhail V. Kozhurin, Paul Leonard, Polina M. Schwarzburd, Mikhail I. Uchitel, Elena A. Zapa-trina, Marina E. Maevskaya, Lyudmila A. Bogdanova. Succinate as a mobilization cue. Cardiometry; Issue 17; November 2020; p.110120; DOI: 10.12710/cardiometry.2020.17.110120; Available from:

“All things are poison, and nothing is without poison, the dosage alone makes it so a thing is not a poison.”

Hippocrates, Paracelsus and Avicenna

Forerunner of the signaling function: small doses of succinate are effective

Practical application of Succinate Containing Compounds (SCC’s) as a biologically active additives and medical substances has shown a surprisingly high effectiveness of small doses of succinate: 1,5÷15,0 mg per kg of body mass (10÷100 micromoles per kilo) [1-11]. The first ever application of ammonium succinate in doses of 0,2 mmoles in composition of a “nerve tonic” has been presented in a Pharmaceutical Рandbook from the XIX century [12]. It is hard to believe that such small concentrations of exogenous succinate introduced via the gastrointestinal tract have really the potential to act as a substrate in Krebs cycle and change the energetic state in mitochondria (MC).

From numerous reports dealing with the performance of isolated mitochondria it is known, that in order to produce an effect on their energy-dependent functions in a meaningful way, we require to oxidize millimoles of succinate, because there is a little similarity between succinate dehydrogenase (SDH) and succinate. Depending on the state of mitochondria, the Km value of succinate for SDH is about 100÷300 micromoles. Healthy normal cells and tissues generally have Km (during the succinate-oxidation activity) by an order of magnitude greater. Such high concentrations of succinate are impossible to reach by supplying small doses of SCC to humans or animals only. Despite that, the effectiveness of small SCC doses has been recorded to be rather significant and long-standing. So, in the absence of intense physical activities, the duration of the changed functioning state of a human, caused by the small doses of SCC, has significantly exceeded the time that it takes for exogenous succinate to metabolize. In particular, in a state of rest, almost complete removal of 13CО2 isotope via exhaled air after application of exogenous succinate enriched with 13C isotope has taken no longer than 60 minutes [13], and the changed psycho-emotional state has been maintained for some hours..

This sort of evidence data is a strong ground to make an assumption that there is a signaling regulatory action produced by a succinate molecule. Initially the assumption was generally formulated as an “up-shift” of the mitochondrial energetics to nervous-endocrine system levels. [14]. With time, more and more of the evidence for possible signaling role of succinate has been accumulated [15,16]. Finally, by the end of the XX and at beginning of the XXI century the search for signaling regulatory roles of succinate has been successfully completed. The regulatory mechanisms have been discovered, which have been found to be highly sensitive to changes of nano- and micromolar concentrations of succinate outside mitochondria and cells.

Extra-mitochondrial succinate is the initiator of the transcriptional hypoxia-inducible factor HIF

In 1995 Gregg L. Semenza together with his colleague Wang Guan discovered transcription factor HIF. Later together with William G. Kaelin Jr. they have identified the role of HIF-1 as an oxygen concentration sensor. In 2019 the researchers have been awarded the Nobel Prize in Physiology or Medicine [The Nobel Prize in Physiology or Medicine 2019: Gregg L. Semenza/ Hypoxia-Inducible Factors in Physiology and Medicine. medicine/2019/semenza/facts/ ] [17,18] It has been established that in case of the sufficient oxygen supply (normoxia) HIF-1 is subjected to hydroxylation with participation of oxygen, 2- oxoglutarate (alpha-keto-glutarate), ascorbate and iron ions. Prolyl-hydroxylated HIF-1 is ubiquitinated followed by fast proteasomal degradation with production of amino acids. An oxygen deficit slows down the HIF1 prolyl-hydroxylase, and the transcription factor is conserved.

It has been detected that a small accumulation of succinate in the cytosol outside the mitochondria (less than 5×10-5М) decelerates prolyl-hydroxylase and thus prevents HIF1 proteolysis. In view of the above, we would like to notice the phenomena of succinate accumulation in the absence of oxygen in mitochondria, tissues and at the level of the organism as a whole [19-22]. Experimental, medical and clinical observations indicate that hypoxia, including any extreme physical activities related to development of hypoxia under physical loading as well as many other diseases, in pathogenesis of which hypoxia, ischemia and inflammations play significant role, are usually accompanied by activation of anaerobic production and even a rise of stationary levels of endogenous succinate [23].

Essentially, an oxygen deficit and/or increase in the concentration of succinate in the cytosol independently facilitate the HIF-1α conservation. As a result, HIF-1α is entering aggregation with HIF-2 forming the HIF dimer. That dimer is an active expressor of the gene complex that is responsible for forming adaptive responses by cells, tissues and an organism to hypoxic effects. The total simultaneous transcription provides for the following translation - the protein synthesis: glucose transporter, glycolysis enzymes, angiogenesis growth factors, erythropoietin, regulators of iron homeostasis and initiators of cell migration, intercellular and inter-organ interactions etc. [17,24,25]. Correspondingly, one of the consequences of the HIF-de-pendent processes launch is an increase in hypoxia resilience and its elimination. This natural adaptive response ensures an exit from anemic and ischemic conditions and adaptation to extreme in their intensity hypoxic stresses. It is vital for survival in cases of an oxygen deficit, since it activates the semi-anaerobic metabolism and maintains the aerobic redox processes due to improvements in oxygen delivery because of the newly grown blood vessels into the zones of impaired blood flow and an increase in the total number of red blood cells in the circulation. Thus an increase in the concentration of extra-mitochondrial succinate is a metabolic alarm signal indicating that hypoxia or ischemic conditions are available. As mentioned above, lack of oxygen is not the only reason for rising levels of endogenous succinate. The latter can be attributed to redox-dependent competitive or allosteric deceleration of succinate dehydrogenase (SDH), upon an effect produced by toxins and SDH-inhibiting drugs, as well as due to genetic enzyme-related abnormalities, particularly in SDHB and SDHD subunits [26,27]. Upon impairment of mitochondrial activity by various type of stresses, released succinate binds to SUCNR1 increasing cell calcium which activates cyclo-oxygenase 1,2 and endothelial nitric oxide synthase [28].

Currently an increase in endogenous succinate and stimulation of the HIF-dependent apparatus is viewed not simply as an adaptive process responsible for the organism’s survival: in some cases it can serve as a sign of pathogenic or inflammatory processes, which may take place in the organism. It is well known that in regions of active neoplasms, as a rule, the partial pressure of oxygen is lower. Furthermore, in some tumors with SDH-abnormal genetics, such as paraganglioma and pheochromocytoma [29], unoxidized succinate is leaving the mitochondria to stabilize the HIF-1α. It should be noted that these are rare tumors, with an overall estimated incidence of 1/300,000. The HIF-di-mer initiated acceleration of glucose uptake, glycolysis and enzyme system activation, which lead to an increase of the NADPH levels and stimulate the biosyntheses in multiplying and maturing cells, promote further tumor development. Formation of new blood vessels may be a factor that accelerates the growth and metastasis of the tumors.

Therefore the HIF initiated activation of the genetic apparatus may not only rise ischemia and hypoxia survival rates, but also promote cancer cell proliferation. This is why the control over the levels of synthesis and breakdown of HIF is one of the key interests to various experts in pharmacology and medicine.

Extracellular succinate is a ligand for the external cellular receptor SUCNR1

In 2004 the following remarkable event in science took place: W. He et al. [30] detected that extracellular succinate in micromolar concentrations can serve as a ligand of the orphaned GPR91 receptor, now known as SUCNR1. This receptor is present on external membranes of cells in many tissues. The signal from an active ligand-receptive complex is initiating an increase in the concentration of intracellular calcium ion as the universal messenger, which regulates the functional cell activity, innumerous amounts of poly-enzyme systems and, in particular, the mobilization of reactions and the catabolism rate. Upon impairment of mitochondrial activity by various type of stresses, released succinate binds to SUCNR1, increasing cell calcium, which activates cyclooxygenase 1,2 and endothelial NO synthase [29]. Currently we have a lot of materials covering the role of the SUCNR1 receptor activation under the influence of increased concentrations of endogenous succinate, as well as high doses of succinate injected into the bloodstream (solutions of 5 - 10 mM of succinate). The discovery of the SUCNR1 receptor, and in particular its activation of the juxtaglomerular apparatus of the kidneys in macula densa, has made it possible to identify another mechanism of arterial pressure increase, which is associated with the renin release due to SUCNR1 activation [ 30].

Thus, succinate, being the intermediary of the Krebs cycle in- and outside the mitochondria, as well as outside the cells, is an important regulatory factor. According to the evidence data provided by scientif- 112 | Cardiometry | Issue 17. November 2020

ic information networks, thousands of publications show the participation and the effects of succinate in a regulatory and adaptive restructuring of the neu-ro-immune-endocrine mega-system functions. This immensely important link between metabolism and the regulatory systems is evident in development of an adaptation to the oxygen deprivation, the mobilization of functional tissue activity and the initiation and pathogenesis of ischemia/reperfusion damages [31], development of the type 2 diabetes, liver pathologies, inflammatory and immune responses. [32, 33, 34]. In a number of pathological states, including carcinogenesis [32, 35], a rise in the stationary levels of endogenous succinate is reported.

Interestingly, the SUCNR1 receptor knockout due to an excess of fats in a diet may have a dichotomous effect: the initial body mass decrease is followed by its repeated increase again, and the same is applicable to the regular receptor activity [36]. As stated above, the progression of hypertension has been predicted already in the first paper discussing the functions of the succinate receptor [30]. Studies in rodents with spontaneous hypertension (SHR line), obesity and diabetes type 2 have shown that an artificial increase in blood succinate leads to an elevation of the mean arterial pressure [37]. However the paper states that such a hypertensive effect has not been found in humans.

The physiological norm of the succinate concentration in blood is about 2÷20 micromoles per liter (μM). Acute hypoxia and hyperglycemia can be accompanied by a succinate release exceeding the norm by 10÷18 times. In case of hyperglycemic spike under the type 2 diabetes this is observed against the background of the initially elevated level of endogenous succinate, which is 1.5-times higher than the norm [33]. Proinflamma-tory stimuli, such as lipopolysaccharide (LPS), interleukin-8, tumor necrosis factor (TNF-α), also initiate a significant rise in the blood succinate concentration [37, 38]. A noticeable short-term spike in the blood succinate contents can be caused by intensive physical activities (~93 μM) [39]. It is worth to mention, that it is incredibly important to differentiate between the transitory short-term elevations of the succinate levels and its stationary concentration alterations, for example, in case of obesity or diabetes (~47 to 125 μM), or in case of some specific changes in intestine microflora [40].

The duration of the blood succinate concentration increase can be of high diagnostic value. Thus, a shortterm elevation in the blood succinate levels under acute myocardial infarction is considered to be an early diagnosis marker, and its rapid decrease is an indication of the favorable MI dynamics. At the same time, a prolonged maintenance of the elevated blood succinate levels after the acute myocardial infarction is, clearly, an adverse symptom of lasting chronic ischemia [41].

The permanent stabilization and activation of the HIF system and the SUCNR1 receptors produced by the elevation of the stationary endogenous succinate concentration has been treated by many researchers as a pathological phenomenon rather than an adaptive one. However, such a simplified interpretation cannot be true. A thorough reference literature analysis has allowed the Norwegian researchers Franco Grimolizzi и Lorena Arranz [42] to describe the diversity of the succinate role as a regulator of metabolism and a participant therein. The analysis they have conducted has determined that the elevation in the blood succinate levels, the activation of the succinate receptor, initiation of the HIF factor transcriptive activity, a change in the SDH activity have extremely ambiguous consequences due to high lability of the regulatory systems and due to high variability of the metabolic systems involving succinate. Therefore, the final result is always dependent on the actual situation available: it may promote either adaptive, physiologically expedient, responses or, by contrast, initiate and stimulate pathogenesis.

Fresh understanding options of a signaling-metabolic role of the succinate molecules have generated anew the interest to the positive effects of succinate. Specifically, an urgent demand to reduce and maintain the transcription activity of HIF has been demonstrated that may be supported both by succinate and some other HIF-1α stabilizers under myocardial infarction against the type 2 diabetes background [43]. Despite the negative anti-inflammatory effect that the endogenous succinate excess has on liver [44], it turns out that these findings are incorrect: in cases of the iron-deficiency anemia in pigs, injections of iron succinate alongside the A and E vitamins has exerted a pronounced healing effect. In addition to the anemia reduction, the condition of the liver has been improving rapidly [45]. Even in cases of diabetes, an inclusion of melatonin in the diet along with a small dose of succinate, despite the risk of an increase in endogenous succinate, is reducing the mitochondrial dysfunction of the liver [46].

It is notable that succinate produced by microbiota or introduced artificially is capable of reducing intes- tine inflammation in mice [47]. Based on the data on the signal effect of exogenous succinate at the hypothalamus level, SCCs have been created to cope with the pathological manifestations of climacteric syndrome in women and reduction of hypogonadism severity in men [48].

What to expect from succinate-containing formulations (SCCs)

The aforementioned evidence demonstrates of how problematic is to predict possible SCC effects of its use a priori. There are many reasons for that, and the most subtle one appears to be the absence of delineation of the endogenous and exogenous succinate effects, mixing of discrete spikes of extra-mitochondrial succinate concentrations with a stationary increase of endogenous succinate in blood that may be caused by a pathology condition. An inadequate extrapolation of evidence data received in vitro to the levels of the whole organism offers also a similar troublemaking way. So, it turns out that the extrapolation of succinate-associated hypertensive potential from animals to humans is an improper method, too [37].

Besides, we should take into account that currently the number of published placebo-controlled, statistically significant pre-clinical and clinical studies of the specific SCC use is extremely limited. Therefore it is almost impossible to a priori predict the possibility of positive or negative effects, which might be produced by newly designed SCCs. Based on the knowledge of aerobic and anaerobic metabolism of succinate in mitochondria of various tissues, the knowledge of gamma-aminobutyric shunt in the nervous system cells, hydroxylation, succination reactions, as well as data on the regulatory and signaling roles of extra-mitochondrial and extracellular succinate allow us to identify and outline the field of the action at best. Since there is a considerable diversity in the SCC family, some statistically significant probabilistic conclusions obtained from the results of the study of one of the types of SCC may serve as certain benchmarks, but not as a guide for the use of other types of SCC in different clinical situations [49]. Despite this, we will try to consider some of the features of the succinate action, based on general biological and pharmacological concepts.

It is well known that small pulse-type influences, like small doses of drugs, play the role of a signal that triggers a certain sequence of biochemical and physiological events. But if the pulse-type effect turns into

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a constant one, and if an inadequately large dose is used instead of a small one, then it can lead to the development of highly unfavorable consequences. This principle of violation of discreteness and an increase in a signal amplitude has been applied to design the most dangerous toxic compounds, such as inhibitors of acetylcholinesterase or monoamine oxidase of catecholamines, etc. to convert the regulatory quantum of acetylcholine or norepinephrine into a permanently acting damaging factor.

Exactly the same differences exist between the effects of short repetitive hypoxic / ischemic acts that perform the adaptive, protective role inherent in pre-and post-hypoxic conditioning and the damaging pathological effects of prolonged hypoxia and ischemia. It is unavoidable to cite an example concerning the progression and treatment of COVID-19, when there is a mixed respiratory, hemic and thrombo-circulatory hypoxia available: under the influence of prolonged hypoxia, a high level of HIF-1α is initiated and maintained for a long time, causing a chain of negative phenomena. Even against this background, hypoxic conditioning sessions that promote short bursts of HIF-1A levels can have a beneficial therapeutic effect [50].

Differences between endogenous and exogenous succinate

So, the first key point that determines the point of view when analyzing the effect of succinate is to understand the differences between the body's responses to a short burst in the endogenous succinate concentration in acute hypoxia/ischemia (as well as after taking SСC) and the pathological conditions that cause a persistent increase in the endogenous succinate levels. Sessions of intermittent hypoxia and peak hypoxic loads are inevitably accompanied by short bursts in the level of extra-mitochondrial and extracellular succinate, which usually play a positive role of the therapeutic and training effects. On the contrary, close to the stationary, the long-term excess of the physiological level and the additional ten-fold concentration increases of extracellular endogenous succinate are characteristic of progressive metabolic syndrome, severe obesity, advanced stages of type 2 diabetes, acute inflammatory infectious processes, a number of types of stable hypertension, progression of ischemic disease, congenital and acquired genetic defects of SDH etc.

Based on the above, obviously we can formulate the following assumption. The succinate signal, which 114 | Cardiometry | Issue 17. November 2020

is small in its duration and amplitude, can provide an adaptive orientation of the body's responses. This phenomenon is fully related to the understanding of the action of SCC application techniques at the level of the whole organism. The fact is that when SCC enters the gastrointestinal tract (GIT), you do not have to expect a significant long-term increase in the level of succinate in the total blood flow and the organs. It is easy to understand by tracing the anatomical and physiological organization of the pathway of a low-molecular substrate, a natural metabolite, when exiting GIT.

Exogenous succinate, upon entering the stomach and then the duodenum and the small intestine, is quickly absorbed by the cells of the mucosa and the abundant microbiota inhabiting the upper GIT segments. Consequently, much less succinate enters the portal vein and is supplied further to the liver than it has been received as a part of the SCC. After metabolic transformations in the liver, even smaller amounts of exogenous substrate enter the lower Vena cava, the right heart, the small circle of blood circulation, and the lungs. After passing through the left heart, the remaining succinate content enters the general bloodstream. In this case, the endothelium of blood vessels, the heart and the lungs oxidize exogenous succinate to CO2 and H2O and include its carbon skeleton into other metabolites. The question of how much of the succinate taken through the GIT enters the great circulatory system has not been studied in detail. It is clear that when taking the described pathway, succinate undergoes a lot of transformations, so that its concentration in peripheral blood and in tissues is very small.

The fate of exogenous succinate entered the bloodstream (introduced directly into the bloodstream) was traced by a radioactive label with simultaneous determination of key intermediates, which included labeled carbon [51]. It turned out that upon expiration of one minute, the concentration of the 14C isotope in blood decreased to 14.9%, 13% of which are not represented by succinate. Consequently, the concentration of exogenous succinate, remaining in the bloodstream, decreased by 7.7 times within 1 minute. In almost all tissues except the brain, the concentration of the isotope label increased rapidly during one - maximum three minutes. And only in the brain, the concentration of 14C increased slowly linearly within 8 minutes (obviously, it is an effect produced by the blood-brain barrier). The distribution of labeled succinate in the organs was very uneven. The highest concentration of succinate was found in the kidneys after a minute; the concentration was reported to be 4 times less in the liver and 10-12 times less in each of the other organs examined. Interestingly, the level of labeled succinate in the heart, despite the high rate of oxidation, was initially 1.5 times higher than in the lungs, the pancreas, or the brain. Metabolic transformations in all organs were quite fast. So, by the end of the observation, which lasted 8 minutes, the total amount of the label in the tissues was 70%, and they were mainly other metabolites. Moreover, in the kidneys, the brain, the liver, and the testicles, 80-95% of 14C was included in other intermediates. Naturally, each tissue had its own peculiarities of metabolism. Thus, in the kidneys, first of all, the content of the label increased in glutamate, aspartate, lactate, and, last of all, in proteins. In the liver, the largest part of 14C was included in glucose and then downwards: in glutamate, malate, lactate and aspartate. In the brain, the largest part of the label was detected in lactate, smaller amounts in glutamate and glucose, malate and aspartate. At the same time, if within the 1st minute the brain contained up to 50% of labeled succinate, then by the 8th minute 47% of 14C were recorded in lactate and 27% were reported to be in glutamate.

The distribution of 14C in organs after intragastric administration of 14C-labeled ammonium succinate in rats is significantly different [13]. At rest, after 30 minutes, the highest concentration of the isotope was found in the liver, that of 14C almost 2 times less in blood plasma and the kidneys and 8 times less in the heart and skeletal muscles, respectively. After physical activity (swimming for 10 minutes with a load of 6% of the body weight in warm water), the rate of excretion of the label with exhaled air in the composition of 14CO2 increased 8-fold as compared to the state of rest, and the concentration of the isotope in the tissues and the blood plasma decreased by 1.5 – 2 times. It is noteworthy that in the blood plasma at the time of slaughter of animals (30 minutes after the introduction of labeled succinate), no more than 1/5 of the total isotope content remaining in the body was detected. Taking into account the data of the above-cited work [51], we believe that at that time the label was in other metabolites, that is, it cannot be a carbon of the succinate itself.

In a human individual at rest, after ingestion of succinate enriched with the non-radioactive isotope 13C in position 2-3, the rate of succinate metabolism is such that almost the entire label is detected in the exhaled carbon dioxide within an hour. Moreover, the conversion of succinate to CO2 is 6-7 times faster than from labeled glucose taken in the same amount [13].

The presented data set gives reason to believe that succinate coming through the GIT cannot provide any long-term high increment in the concentration of succinate either in blood or in tissues. You may expect a small burst in the concentration of succinate for no longer than a dozen minutes, and, consequently, a shortterm signaling effect of exogenous succinate only.

It is clear that small doses of exogenous succinate received as a part of the SCC cannot be compared with the array of formed and oxidized endogenous succinate. Let us remind that in the tissues of the body with a balanced diet of carbohydrates, fats and protein per day at the state of rest (with an energy requirement of less than 2000 kcal), about 450 g of succinate, that is, more than 3.5 moles, can be formed and oxidized in MC. In the absence of hypoxic conditions and disorders in the Krebs cycle, nothing should theoretically enter the bloodstream from this huge amount of endogenous succinate metabolized in MC. It is not surprising that under the physiological conditions in humans at rest, the current level of succinate in blood is 2-20 μm. This concentration seems to reflect the integral result of the contributions of the proper macroorganism and the GIT microbiota. In rodents, whose nutrition pattern differs from that of humans in a high content of plant fibers as a succinatogenic diet, producing a more hypoxic metabolism and a richer microflora in the GIT, the level of succinate in blood is on average 2 times higher than that in humans. Perhaps this is why the parenteral administration of 5 μm of exogenous sodium succinate under pathological conditions was able to cause a rise in arterial pressure (AP) in rodents that was not observed in the same situation in humans [37]. Obviously, an extrapolation of the animal experiment results to the human level is not always successful, and different succinate salts cannot be evaluated as mere succinate without taking into account the role of the cation.

Interestingly, in our studies, ammonium succinate administered through the stomach at a dose of 4 μm did not cause an increase in AP even in spontaneously hypertensive rats. There was also no increase in AP after a single or course administration of SCC containing ammonium succinate during 2 months in the examined patients in 4 placebo-controlled studies

• [5 2,53]. Our study illustrates that the expectation of a hypertensive effect is not justified.

The evidence for the dangerous role of exogenous succinate as an initiator of increased intestinal epithelial permeability and the development of inflammation in the GIT mucosa in pigs, which were given inadequately high doses of succinate, is highly critical. Pigs were watered for several weeks not with water, but with 1% succinate, that is, 80 millimolar succinate solution. The animals received about 1 g of succinate per kg of body weight daily. In the same study [54], negative data were obtained in vitro after incubation of tissue preparations in the presence of 5 μm succinate. It was absolutely not taken into account that the concentration of extracellular succinate in pigs is close to that in humans, that is, in the area of ten micromoles. It should be remembered that in the XIX century and at the present time in the composition of SCC nutraceuticals and oral medications, it is recommended to take a single dose or a course of 0.2 to 3 mmol of succinate [12, 6-10, 52,53]. Consequently, in the cited study [54], there is at least a 100-fold excess of the dietary and therapeutic doses. On the one hand, such "horror stories” can only surprise us. On the other hand, the presence of a clearly manifested effect that differs from the placebo effect [52, 53], when ingesting small amounts of SCC, in which only the signaling effect of almost subthreshold succinate concentrations can be manifested, is really amazing.

Conclusion: nutraceutical succinate models a discrete signal of the interrelation between metabolism and a variety of the body’s regulatory systems

Succinic acid, being an intermediate of the Krebs cycle and the end product of anaerobic transformations in MC, can be formed to a much lesser extent in the GABA shunt from succinic semialdehyde, in the glyoxalate cycle, and in hydroxylation reactions involving a- ketoglutarate. It is important to take into account that succinate is a signaling molecule that plays the role of one of the key agents in the relationship between tissue metabolism and the regulatory systems of intracellular, intercellular and distant systemic signaling in humans and animals. In addition, succinate is involved in the interaction of the macroorganism and the GIT microbiota. This fact raised the question of how to evaluate the role of succinate produced by the GIT microbiota: is it a friendly relationship or a hostile 116 | Cardiometry | Issue 17. November 2020

action of the microflora against the macroorganism [55]. The microbiota may be responsible for maintaining and changing the level of endogenous succinate in blood, especially with a diet rich in plant fibers [56] or with shifts in the composition of the microbiota induced by systematic intense physical activity [57]. Thus, succinate acts as an active agent that combines metabolism and the regulation of the functions of an integral holo-organism. Along with other factors, this circumstance must be taken into account when using antimicrobial agents, organizing a diet, changing the regime and intensity of loads.

The deepening and expansion of the knowledge in the field of the succinate-dependent regulation has opened up new ways and identified new targets and goals for the treatment of metabolic syndrome and a number of pathologies in the nervous, immune and endocrine systems. We should note that in recent years, the signaling regulatory function has been detected not only for succinate, but also for other common or pathology-related metabolites in catabolism and metabolism plasticity [58, 59, 60].

The condition for producing a critical, constructive, understanding of the phenomenology and the regulatory mechanisms associated with succinate is a clear distinction between its endogenous and exogenous sources, taking into account the active concentrations and the informational value of the duration of various endogenous and external succinate signals. The discreteness or the permanence of the succinate exposure, differences in the amplitude and the current background determine the physiology, adaptability, or, on the contrary, pathogenicity of succinate signaling at all levels: from mitochondria, cell cytosol to intercellular and inter-organ communications. Due to the initial delivery of the SCC to the stomach, intestines and GIT microbiota, passing through the metabolic "obstacles" of the liver, the heart and the lungs, and due to the extremely high metabolic rate, the succinate signal is short and has small amplitude of an increment of the concentration in blood and tissues.

Short bursts of the endogenous succinate concentration have an adaptive effect, for example, after sessions of intermittent hypoxia and hypoxic post-conditioning, rapid recovery from ischemia, and transient extreme glucose load. A long-term increase in the level of endogenous succinate, on the contrary, reflects the presence of systemic metabolic and / or genetic pathology: obesity, insulin resistance, diabetes, hyper- tension, chronic general and local circulatory disorders, prolonged ischemia of various genesis, and the progression of neoplasms. A permanently elevated level of succinate outside MC and outside the cells also has its negative consequences, as well as the transformation of the physiological pulse-type signal of regulation into a continuous bombardment of the central and peripheral nervous system, excitable organs like the heart, skeletal muscles, intestines, and neuroendocrine apparatus with stimulating agents. This transformation of the pulsed quantum regulation signals into constant high-amplitude ones at any level triggers an execution of the sequence covering hyperfunction, hypertrophy, dystrophy, and involution of target organs.

The available materials allow us to believe that the use of SCC promotes the launch and adaptive mobilization of functions similar to natural hypoxic conditioning due to the fact that the generation of a low-amplitude short succinate signal is provided. It is surprising that even against a disturbed, pathologically elevated background of endogenous succinate, short small signals can have a preventive and curative effect.

In any situation, it should be borne in mind the importance of determining the boundary conditions for the use of exogenous succinate and succinathogen-ic agents to achieve the desired goal. Specifications of the state of an object and examinations of the actual situations with the use of adequate methods of monitoring and control are required. It is obvious that single doses and course prescriptions of SCC, succinato-genic products, agonists, antagonists or inhibitors of the succinate-dependent regulatory systems require further development of clear-cut criteria for assessing effectiveness and safety, indications, ranked sampling of human individuals and animals, and in an ideal case personification. In other words, general ideas can be a base expanded with the specific knowledge of the applicability of different succinate-containing agents and of the body's responses to them, even if we deal with different salts of succinate.

At least, a priori statements about "the absolute safety" of dietary supplements containing natural metabolites cannot be treated as weighty arguments. In recent years, for many metabolites: substrates, intermediates, final products of biochemical transformations, as well as their derivatives, have been reported to have a signaling regulatory function. This information encourages classifying the previously obtained data and conventional approaches aimed at studying responses at the level of the regulatory, in particular molecular genetics, systems. It appears that the time is near when natural substances will be tested according to the relevant NOAEL criteria established to identify ‘the greatest concentration or amount of a substance found by experiment or observation which causes no detectable adverse alteration of morphology, functional capacity, growth, development or life span of the target organism under defined conditions of exposure’. So far this standard has been applied only to medical drugs, but its scope is now expanding. Only specific research work is capable of definitely answering questions whether benefits or dangers might be expected in case of the use of a recommended natural substance or composition.

Statement on ethical issues

Research involving people and/or animals is in full compliance with current national and international ethical standards.

Conflict of interest

None declared.

Author contributions

The authors read the ICMJE criteria for authorship and approved the final manuscript.