Lactate as a Metabolite and a Regulator in the Central Nervous System

More than two hundred years after its discovery, lactate still remains an intriguing molecule. Considered for a long time as a waste product of metabolism and the culprit behind muscular fatigue, it was then recognized as an important fuel for many cells. In particular, in the nervous system, it has been proposed that lactate, released by astrocytes in response to neuronal activation, is taken up by neurons, oxidized to pyruvate and used for synthesizing acetyl-CoA to be used for the tricarboxylic acid cycle. More recently, in addition to this metabolic role, the discovery of a speci c receptor prompted a reconsideration of its role, and lactate is now seen as a sort of hormone, even involved in processes as complex as memory formation and neuroprotection. As a matter of fact, exercise offers many bene ts for our organisms, and seems to delay brain aging and neurodegeneration. Now, exercise induces the production and release of lactate into the blood which can reach the liver, the heart, and also the brain. Can lactate be a bene cial molecule produced during exercise, and offer neuroprotection? In this review, we summarize what we have known on lactate, discussing the roles that have been attributed to this molecule over time. Keywords: lactic acid; brain metabolism; lactate transporters; blood-brain barrier; lactate receptors; exercise and lactate 1. Introduction Carl Wilhelm Scheele, an 18th

also the brain. Can lactate be a bene cial molecule produced during exercise, and offer neuroprotection? In this review, we summarize what we have known on lactate, discussing the roles that have been attributed to this molecule over time. Keywords: lactic acid; brain metabolism; lactate transporters; blood-brain barrier; lactate receptors; exercise and lactate 1. Introduction Carl Wilhelm Scheele, an 18th century Swedish chemist, also known for his role in the discovery of oxygen, puri ed many organic acids, among which lactic acid (LA). About one hundred years later, in their study on the regulation of the blood supply to the brain, Roy and Sherrington reported that cerebral anemia, experimentally obtained by closing the carotid and vertebral arteries, was immediately accompanied by an acid reaction in the cortex; then, after reopening the cerebral vessels, the acidity gradually disappeared. The Authors attributed the observed effects to the formation of (ethylidene) lactic acid [1]. In the following years, it was proposed that LA increased during muscle exercise because of an oxygen debt, determined by the high energy requirement of contraction [2–4]. In glycolysis, indeed, glucose is broken down to two molecules of pyruvate; pyruvate then either enters the mitochondria, where it is further oxidized in the tricarboxylic acid (TCA) cycle, or is reversibly reduced by NADH(H + )-dependent lactic dehydrogenase (LDH) to lactic acid, in order to regenerate NAD + for glycolysis. Shortage of oxygen, by slowing down mitochondrial oxidative Int. J. Mol. Sci.2016,17, 1450; doi:10.3390/ijms17091450

Int. J. Mol. Sci.2016,17, 1450 2 of 20 phosphorylation, and hence TCA cycle, causes an increase of lactate production. In order to limit cell acidi cation, both lactate and protons then exit cells. By the end of the fties, it was clear that hypoglycaemia, by limiting the supply to the brain of its preferred metabolic substrate (i.e., glucose), could reduce cell respiration/energy production and hence brain electric activity. It was also clear that, upon electrical stimulation, the metabolic activity of the brain could be increased and, as a consequence, more glucose and oxygen were taken from the blood, with a parallel increase of carbon dioxide and lactate released into the blood [5]. Moreover, electrical stimulation of brain tissue rendered it more susceptible to a fall in glucose level, and the same effect was noticed for the aerobic accumulation of LA [6]. These observations suggested that neuronal activity needs to be coupled to an ef cient uptake of oxygen and metabolites from the circulation, and that the blood ow itself should be modulated locally in response to variations of neuronal activity [7]. In the following decades, the possibility emerged that cells in the brain itself, namely astrocytes, can transfer to neurons molecules, such as glucose and/or lactate, to sustain increased energy requirements, both in physiological and pathological conditions. Here, we'll discuss how ideas on the production and utilization in the brain of LA have changed over time from identi cation of lactate as a metabolic product of glucose metabolism to acknowledgement of its regulatory functions. 2. Exercise, Lactate Production in the Periphery and Fatigue 2.1. Exercise and Lactate Production In muscle cells, during intense exercise, ATP is mainly generated from blood glucose and muscle glycogen to fuel glycolysis. This latter pathway is still considered the fastest, but produces less ATP compared to phosphagen systems and to the cellular respiration (oxidative phosphorylation, OXPHOS) [8]. ATP, the fuel used for contraction, is usually present in cells at a concentration of 8 mmol/kg of wet weight of muscle [9]. On the other hand, accumulation of pyruvate, not used for OXPHOS, should inhibit glycolysis because

is still considered the fastest, but produces less ATP compared to phosphagen systems and to the cellular respiration (oxidative phosphorylation, OXPHOS) [8]. ATP, the fuel used for contraction, is usually present in cells at a concentration of 8 mmol/kg of wet weight of muscle [9]. On the other hand, accumulation of pyruvate, not used for OXPHOS, should inhibit glycolysis because of shortage of NAD + ; to overcome this situation, in anaerobic conditions (hypoxia), a large part of pyruvate is reduced to lactate by lactate dehydrogenase enzyme, thus oxidizing NADH(H + ) back to NAD + . Noteworthy, during intense physical activity, lactic acid, that is a strong organic acid, mainly exists, like pyruvate, as an anion at the human body pH values. In other words, due to its pKa (3.86), lactic acid is physiologically deprotonated and exists as lactate and H + [10]. In addition, lactate can be also considered an aerobic metabolite, usable by skeletal muscles and by the heart when the oxygen availability is adequate, and it may contribute to acetyl-CoA formation [11]. A large part of muscle-derived lactate is transported to the liver where it is used to synthesize glucose through gluconeogenesis; glucose then enters the blood ow and comes to the muscles to be used as a metabolite in glycolysis (Cori's cycle). In the last decade, it has been discovered that adipocytes can also produce lactate under anaerobic circumstances. It has been suggested that, in order to counteract O2shortage, adipocytes switch to glycolytic metabolism, and to avoid a dramatic intracellular pH decrease, protons are removed from the cell thanks to proton-linked monocarboxylate transporters (MCTs; isoforms 1 and 4; see next section) [12]. The total amount of lactate produced by adipocytes depends on the fat mass: it is indeed higher in obese subjects than in normal-weight individuals. A large part of lactate produced from active muscles is released and taken up by exercising and non-exercising muscles, heart and brain, to be oxidized and used as a fuel [13]. More than 30 years ago, Brooks introduced the concept of lactate shuttle through which lactate might be carried

it is indeed higher in obese subjects than in normal-weight individuals. A large part of lactate produced from active muscles is released and taken up by exercising and non-exercising muscles, heart and brain, to be oxidized and used as a fuel [13]. More than 30 years ago, Brooks introduced the concept of lactate shuttle through which lactate might be carried into mitochondria or peroxisomes, through MCTs, to be reoxidized, or even out into the bloodstream to be taken up from other organs which could use it as a fuel [14]. In sports medicine, the highest level of physical effort that the body can sustain without accumulating lactate and hydrogen ions (H + ) in blood and muscles is called anaerobic threshold or lactate threshold; this threshold also corresponds to the intensity/duration of physical activity that the energy metabolism supports without switching from aerobic to anaerobic. Lactate threshold provides a good tness level and is considered

Int. J. Mol. Sci.2016,17, 1450 3 of 20 the best performance predictor. Intense training increases lactate threshold and, nally, increases performance [15]. Hypoxia can be induced by intense exercise as well as by altitude exposure (2300–5700 m); the latter induces skeletal muscle adaptations, such as transport of bicarbonate, hydrogen ions, and lactate, all of which are nely regulated. Oxygen shortage stimulates glycolysis and can increase pyruvate availability. This may be used for further oxidation in mitochondria or to increase lactate production. As a result of hypoxia, there is a higher amount of lactate production for a given workload. Muscular energy comes more from carbohydrates than fatty acids. Altitude-induced hypoxia leads to the lactate paradox, de ned as “reduced blood lactate concentrations during submaximal and maximal exercise following acclimatization to high altitude” [16]. An interesting hypothesis is that altitude acclimatization may be due to a reduced “muscle recruitment” probably aimed at “protecting” cerebral oxygenation; in other words, the paradox of lactate, observed in chronic hypoxia, should be due to reduced muscle recruitment imposed by the central nervous system, that reduces the transport of O2 to muscles and muscle contraction to protect itself from dangerous hypoxia [16,17]. 2.2. Fatigue A parallel interesting matter concerns “fatigue”, a condition of rapid deterioration of contractile function, caused by intense exercise. Keeping ATP levels is essential in carrying out the physiological functions of the body. Muscle fatigue is possibly necessary to avoid that ATP range falls below critical or irreversible levels, and it is logical to assume a causal relationship between anaerobic metabolism and muscle fatigue [18]. The shift from aerobic to anaerobic metabolism is probably responsible for the sudden increase in lactate levels in blood and, even before, at the intracellular level, for metabolic acidosis [19]. Acidosis seems to impair muscle contraction, leading to fatigue and, ultimately, to cessation of exercise. The uncomfortable feelings associated with acidosis has long been associated with the pain (now commonly referred to as delayed onset muscle soreness, or DOMS) of the days after intense exercise. Lactate production through the glycolytic pathway is associated with hydrogen ions (H + )

[19]. Acidosis seems to impair muscle contraction, leading to fatigue and, ultimately, to cessation of exercise. The uncomfortable feelings associated with acidosis has long been associated with the pain (now commonly referred to as delayed onset muscle soreness, or DOMS) of the days after intense exercise. Lactate production through the glycolytic pathway is associated with hydrogen ions (H + ) production: in particular, two ions are released if glycolysis uses blood glucose, while one ion is released if glycogen is used [20]. Thus, lactate is not necessarily the source of the H + increase, which might indeed derive from glycolysis [21]. Historically, the accumulation of lactic acid in muscles has been suggested as the main cause of muscle fatigue. Lactate and H + ions are produced in muscles during intense exercise, and in humans, the intracellular lactate concentration may reach 30 mM or more. Nevertheless, the intracellular pH only decreases of approximately 0.5 units, due to the body buffering capacity. In these conditions, an increase of extracellular osmolarity should be observed, which causes a water movement out of the muscle bers, and thereby an increase of the intracellular ionic strength. The overall result should be an inhibitory effect on force production [22,23]. On the other hand, acidi cation per se seems to have a low effect on the reduced ber shortening velocity linked to fatigue. Lactic acid production plays indeed a metabolic buffering action rather than determining the onset of acidosis. As a matter of fact, the reaction catalyzed by lactate dehydrogenase uses two electrons and one proton from NADH and a second proton from the cytosol to reduce pyruvate to lactate [24]. In summary, the muscle lactate production is essential for the buffering effect as well as to regenerate NAD + , at the same time serving to remove the extra pyruvate [9]. A further mechanism by which intracellular acidosis might induce fatigue is through inhibition of key enzymes of glycogenolysis and glycolysis, such as glycogen phosphorylase and phosphofructokinase, both of which are inhibited by low pH, at least in vitro [25]. Another system responsible for ATP synthesis in

NAD + , at the same time serving to remove the extra pyruvate [9]. A further mechanism by which intracellular acidosis might induce fatigue is through inhibition of key enzymes of glycogenolysis and glycolysis, such as glycogen phosphorylase and phosphofructokinase, both of which are inhibited by low pH, at least in vitro [25]. Another system responsible for ATP synthesis in working muscles involves the phosphagens. During intense exercise, the main substrate used is creatine phosphate (CrP). The reaction catalyzed by creatine kinase uses protons (H + ), thus causing a faint alkalization of the muscle cell. The effect of this reaction is to counteract the slight acidosis induced by glycolysis and lactic acid formation [26]. Muscle contraction also causes an increase of phosphate (Pi) levels, which has a direct effect on myo brils and/or an effect on the pathway of excitation/contraction of muscle cells [27].

Int. J. Mol. Sci.2016,17, 1450 4 of 20 Several studies suggest that an elevated muscle [H + ] as well as high [Pi] could impair muscle function by reducing (i) the transition of mio brils to high cross-bridged states; (ii) the maximal shortening velocity; (iii) the glycolytic rate; (iv) cross-bridging activation, by competitively inhibiting Ca 2+ binding to troponin C [4,28,29]. It is not yet clear which effect has the predominant role in fatigue; probably, all the mentioned factors are involved, and play a synergistic effect, thus leading to impaired contraction and muscle strength. 3. Lactate Uptake across the Blood-Brain Barrier and Monocarboxylate Carriers (MCTs) 3.1. Lactate Can Cross the Blood-Brain Barrier (BBB) During the eighties, the idea that lactic acid (LA) was only a dead-end waste product of anaerobic metabolism began being challenged by many converging observations [4]. First of all, as mentioned, it was realized that LA can be shuttled both between and within the cells. The most relevant output of LA from cells was bound to sustained exercise. This process was de ned by Brooks [14] as the “lactate shuttle”. LA is indeed released from muscles during short-term exercise, and even from muscles at rest, during recovery from short-term exercise, and during long-lasting exercise. However, LA can be taken up from blood into either resting or slowly working muscles [4]. Actually, in the same muscle, glycolytic bers can produce and release LA which can be, at least in part, taken up by surrounding oxidative bers and again converted to pyruvate for further aerobic oxidization. As we will discuss later, a relevant case of intercellular lactate shuttle should be that established among glial cells (especially astrocytes) and neurons, which could be considered one of the many aspects of the glial–neuronal metabolic coupling. In addition to intercellular lactate shuttling, intra-cellular shuttling has been also suggested. In particular, muscular mitochondria were reported to contain a monocarboxylate transporter (MCT1, see below), able to allow LA entrance, as well as lactic dehydrogenase, which converts lactate back to pyruvate. This reaction also transfers electrons to NAD + , thus also shuttling electrons from

the glial–neuronal metabolic coupling. In addition to intercellular lactate shuttling, intra-cellular shuttling has been also suggested. In particular, muscular mitochondria were reported to contain a monocarboxylate transporter (MCT1, see below), able to allow LA entrance, as well as lactic dehydrogenase, which converts lactate back to pyruvate. This reaction also transfers electrons to NAD + , thus also shuttling electrons from the cytosol to the mitochondrion [4,30,31]. A further indication that lactate is not only a waste product of anaerobic metabolism, produced in conditions of oxygen shortage, but instead a potentially useful fuel, came from the observation that the blood brain barrier (BBB) can be crossed by lactate. The brain is indeed isolated and protected from compounds circulating in the blood by a layer of brain capillary endothelial cells (BCECs) with peculiar properties [32]. BCECs are sealed together by tight junctions (TJs) which block para-cellular uxes of molecules. Thus, in order to enter the brain, molecules have to be either soluble in plasma membrane lipids (and not actively extruded by ATP-dependent transporters: ABC transporters), or to be recognized by transporters, able to mediate trans-cellular crossing of the BCEC layer [32–35]. In addition, to ensure trans-cellular transport, a synergic cooperation between the two membranes of endothelial cells is required [36]. 3.2. The Monocarboxylate Carriers (MCTs) Lactate entrance into the brain is mediated by BBB transporters which belong to the monocarboxylate carrier (MCT) family, and also transport other monocarboxylic acids, such as pyruvate and ketone bodies. MCTs form a family of 14 transmembrane proteins [37,38], which belong to the solute carrier family (SLC16), and for many of which endogenous functions and substrates still await to be discovered. Actually, lactate transport across the cell plasma membranes of the body can use either sodium-coupled transporters (SMCTs) or proton-coupled monocarboxylate transporters (MCTs) [13]. In the brain, lactate transport involves MCTs. In particular, four carriers (MCTs 1–4) have been characterized which catalyze bidirectional, electroneutral 1:1 co-transport of protons and monocarboxylic acids [13,37–40]. All of them can transport lactate, with different af nities, the one with the highest af nity being MCT2 (Km: about 0.7 mM). MCT1 is

(SMCTs) or proton-coupled monocarboxylate transporters (MCTs) [13]. In the brain, lactate transport involves MCTs. In particular, four carriers (MCTs 1–4) have been characterized which catalyze bidirectional, electroneutral 1:1 co-transport of protons and monocarboxylic acids [13,37–40]. All of them can transport lactate, with different af nities, the one with the highest af nity being MCT2 (Km: about 0.7 mM). MCT1 is expressed in a variety of human tissues,

Int. J. Mol. Sci.2016,17, 1450 5 of 20 including brain, and is involved in brain uptake of lactate across the BBB both in mice and humans [41]. MCT2, rst reported to be expressed in astrocytes, has been then demonstrated to be primarily present in neurons, and, more precisely, in postsynaptic elements of glutamatergic synapses, where it has been suggested to provide increased supply of lactate “as energy fuel” during neuronal increased activity [39]. MCT3 is preferentially expressed in the basolateral membrane of the retinal pigment epithelium (RPE), and plays an important role in regulating pH and lactate concentration in the outer retina [42]. Finally, MCT4 (Km: about 35 mM) seems to be restricted to the plasma membrane of astrocytes, where it localizes to both perisynaptic processes and perivascular endfeet [39]. The different localization of the MCTs in the brain suggests that each species might be involved in different aspects of lactate metabolism, possibly involving differences in the preferential direction of inter-cellular lactate transport. The role of these transporters might change under different conditions. It has been reported, for example, that oxygen- and glucose-deprivation (OGD) increases cell death in cultured neurons, and up-regulates expression of MCT4 in astrocytes. If, however, cell cultures are exposed to MCT4- or MCT2-speci c siRNAs, thus signi cantly reducing their expression, neuronal cell death increases, unless lactate is added to the culture medium before OGD [43]. Moreover, expression of MCTs has been found to be perturbed in some pathological conditions, such as epilepsy [41]. The MCTs have different Km for lactate, thus probably contributing to the speci c tendency of each cell type in a tissue to transport lactate out of or into the cell. It is, however, to be pointed out that the proton gradient is also important [39]. Interestingly, membrane localization and activity of MCTs also require accessory proteins, such as neuroplastin, basigin and embigin [44–46], that chaperone their transport to the membrane as well as their function. Recently, MCT1, MCT2, and MCT4 have been shown to be expressed also in the peripheral nervous system (PNS), with different cell- and domain-distribution: myelinating Schwann cells

This review discusses the evolving roles of lactate in the nervous system.