Tracing the lactate shuttle to the mitochondrial reticulum

Isotope tracer infusion studies employing lactate, glucose, glycerol, and fatty acid isotope tracers were central to the deduction and demonstration of the Lactate Shuttle at the whole-body level. In concert with the ability to perform tissue metabolite concentration measurements, as well as determinations of unidirectional and net metabolite exchanges by means of arterial–venous difference (a-v) and blood flow measurements across tissue beds including skeletal muscle, the heart and the brain, lactate shuttling within organs and tissues was made evident. From an extensive body of work on men and women, resting or exercising, before or after endurance training, at sea level or high altitude, we now know that Organ–Organ, Cell–Cell, and Intracellular Lactate Shuttles operate continuously. By means of lactate shuttling, fuel-energy substrates can be exchanged between producer (driver) cells, such as those in skeletal muscle, and consumer (recipient) cells, such as those in the brain, heart, muscle, liver and kidneys. Within tissues, lactate can be exchanged between white and red fibers within a muscle bed and between astrocytes and neurons in the brain. Within cells, lactate can be exchanged between the cytosol and mitochondria and between the cytosol and peroxisomes. Lactate shuttling between driver and recipient cells depends on concentration gradients created by the mitochondrial respiratory apparatus in recipient cells for oxidative disposal of lactate.


INTRODUCTION
The results of isotope tracer studies at the whole-body and tissue levels have clarified longstanding issues concerning metabolic regulation. In particular, the role of the mitochondrial reticulum 1,2 in lactate disposal has been identified. The reticulum lowers cellular [lactate] by oxidative disposal, thus acting to develop the concentration gradients necessary for lactate fluxes across organ, tissue, and cellular compartments. Similar to other solutes, gases, and entities in physics and physiology, lactate moves from areas of high to areas of low concentrations. Hence, lactate-producing cells, organs, and tissues with high lactate levels drive carbon flux to disposal (recipient) sites with low lactate levels. In this scenario, the mitochondrial reticulum, in which lactate is disposed of via oxidation, creates the driving force for shuttling lactate around the body and within organs, tissues and cells [3][4][5] . Key features of the process involve the mitochondrial (m) Lactate Oxidation Complex (mLOC), which contains mitochondrial lactate dehydrogenase (mLDH), a monocarboxylate (lactate) transporter (mMCT), a scaffolding protein (CD147) and cytochrome oxidase (COx). However, within the context of this special issue on the use of isotopic tracers to study metabolism, the results of lactate tracer studies directly led to the discovery of the mLOC for lactate shuttling to occur in vivo. We begin with a discussion of mLDH.
Similar to the concept of an mLOC, discussions on the role of mLDH in intermediary metabolism have endured a long and winding history 3,5,6 , perhaps a path not unusual in the history of science 3,7,8 . Because of the fundamental importance of mLDH in physiology and metabolism, revisiting the history of discoveries is warranted at the outset of this paper. The presence of mLDH is essential to explain the role of lactate in affecting lactate flux and ultimately the regulation of energy substrate partitioning in vivo 3,5,9 .
Background knowledge Today, we know that some types of facultative cells can be cultured with lactate as a preferred fuel because their mitochondrial reticulae consume and oxidize lactate directly without conversion to pyruvate in the cytosol. For instance, mitochondria of yeast (Saccharomyces cerevisiae) contain Flavocytochrome b 2 , a lactate-cytochrome c oxidoreductase 10 that couples lactate dehydrogenation to the reduction of cytochrome c 11 . In fact, the association between cytochrome b 2 and LDH in yeast can be traced to the 1940s 12 . A similar phenomenon occurs in mammalian muscle [13][14][15][16][17][18][19] , in persons resting at the sea level, and even in men exercising at sea level or at an altitude of 4300 m 20,21 . Technological limitations, such as the availability of isotope tracer methodology, the ability to catheterize veins and arteries for continual (a-v) measurements, the ability to determine tissue and systemic blood flow rates, and mass spectrometry for turnover analyses, made such data unobtainable in the 1920s. Consequently, longstanding ideas on the role of lactate in physiology originated on the basis of incomplete data on isolated, nonperfused, nonoxygenated amphibian muscles made to contract continually in ways not intended by nature [22][23][24][25][26][27] . More recently, with the advent of isotope tracers and nuclear magnetic resonance (NMR) spectroscopy, we now know that contractions stimulate glycolysis in muscle independent of O 2 availability [28][29][30][31][32][33] and that lactate is oxidized in working skeletal 31,34,35 and cardiac 33,[36][37][38][39] muscles.

MITOCHONDRIAL LACTATE OXIDATION (THE MIND'S EYE VIEW OF POLYPHEMUS)
Contemporary textbooks of biochemistry and physiology abound the 19th-century concepts that metabolism is either "anaerobic" (without O 2 ) or "aerobic" (with O 2 ) 40 . Glycolytic flux from glucose and glycogen is typically depicted in textbooks as progressing to pyruvate and then to the tricarboxylic acid (TCA) cycle. However, if oxygen is absent, textbooks assert that glycolysis progresses to lactate [41][42][43] . This is a convenient motif, typically copied by one textbook author from another and then through serial editions of texts. Amazingly, some textbook authors who advanced the idea of lactate production due to oxygen limitation were biochemists who worked with cells in high-glucose-containing culture media under fully aerobic conditions of one atmosphere pressure in which the partial pressure of oxygen was at least 50% greater than in the arterial blood of individuals at sea-level altitudes. Because the maintenance of cells in such preparations required daily changing of the incubation media to maintain high [glucose], low [lactate], and physiological pH, the observations should have informed the investigators that lactate was produced under fully aerobic conditions. Contemporaneously, physiologists measured lactate [L] to pyruvate [P] concentration ratios (L/P) of 10 in muscles and blood in resting mammals, including humans, and further observed the L/P to rise more than 100 during submaximal exercise 44 . However, did any of the textbook authors ever look to determine whether isolated mitochondria oxidize lactate? For many textbook authors [41][42][43] , the answer is "no." However, for a few others, the answer is "yes" 45,46 . Moreover, some investigators also looked for the presence of a mitochondrial monocarboxylate (lactate) transporter (mMCT) and a lactate dehydrogenase (mLDH) enzyme. Unfortunately, while some attempts failed [47][48][49] , fortunately, other attempts to observe mitochondrial lactate oxidation and the presence of mLDH and mMCT were successful 13,[50][51][52][53][54][55] . Importantly, lactate oxidation in human muscle mitochondrial preparations was observed 56 . Regrettably, for a time, positive results were overlooked or castigated as being "controversial" and dismissed for failing to fit with established paradigms of metabolic regulation. However, now that the MitoCarta (https:// www.broadinstitute.org/mitocarta/mitocarta30-inventorymammalian-mitochondrial-proteins-and-pathways) 57 and MitoMiner (https://mitominer.mrc-mbu.cam.ac.uk/release-4.0/begin.do) databases have been published, mLDH is a recognized constituent of the mitochondrial proteome. Acknowledging that lactate, not pyruvate, is the main gluconeogenic precursor and carbohydrate energy source shared at the organismal and tissue levels upsets archaic concepts of the organization of intermediary metabolism. Nevertheless, despite the evidence, in the manner of Polyphemus, some 58,59 have ignored the literature and databases on mLDH and mMCT and their roles in mitochondrial lactate oxidation 60 .
The lactate:pyruvate affair Regrettably and inexplicably, thus far, as the use of isotope tracers to study lactate metabolism is concerned, the literature has been muddled, creating a "Lactate:Pyruvate Affair" that needed to be corrected 60 . Specifically, there has been controversy over whether lactate tracers measure lactate (L) or pyruvate (P) turnover. Despite a series of analytical errors, the use of inappropriate tissue and animal models, a failure to consider L and P pool sizes in modeling results, inappropriate tracer and blood sampling sites, a failure to anticipate roles of heart and lung parenchyma on L ⇔ P interactions, and a failure to appreciate results from magnetic resonance spectroscopy (MRS) and immunocytochemistry 61,62 , it is clear that carbon-labeled lactate tracers can be used to quantitate lactate fluxes in situ and in vivo.

The early twentieth century
In the early literature of the 20th century, there were several threads to a contemporary understanding of lactate production and oxidation during postprandial and exercise conditions. Among those threads were the findings of Fletcher and Hopkins, who showed that lactic acid disappeared when frog muscles fatigued by electrical stimulation were placed in oxygen-rich environments 63 . Albeit in another field of endeavor, cancer research, another thread was the finding of Warburg that lactate was produced by fully oxygenated cancer cells 64 . Nonetheless, while there were data from a reputable investigator (Nobel Laureate) that lactate could be produced under fully aerobic conditions and that oxidative removal of lactate could be accomplished without reconversion to glucose, lactate and lactic acid in cell metabolism were misconstrued as waste products of oxygen-limited metabolism and fatigue agents early in the 20th century [65][66][67] .
Most importantly, several sets of findings in the late 20th century led to the realization of oxidative lactate disposal. Among these sets of findings was evidence that isolated mitochondrial preparations could oxidize lactate in vitro 13,54 and that mitochondria contained the enzymatic apparatus to oxidize lactate 13,53,68 . Importantly, isotope tracer studies showed oxidative lactate disposal in mammals and humans in vivo [69][70][71][72][73] . Evidence obtained on individuals studied in vivo is elegantly supported by the results of studies using MRS technology 28,33,34,74 . These important sets of findings are addressed sequentially.

OVERLOOKED FINDINGS
The first investigator to evaluate mitochondrial lactate metabolism was Mario Umberto Dianzani (1925Dianzani ( -2014, who, in 1951, published a paper, translation of which is "Distribution of lactic acid oxidase in liver and kidney cells of normal rats and rats with fatty degeneration of the liver" 55 . A translation of a section on p. 182 is "In conclusion, the [cellular] lactic acid-oxidizing systems are localized 80 to 100% in the mitochondria." Although Dianzani had a prolific career in hepatic toxicology and only recently retired from the Department of Experimental Medicine and Oncology, University of Turin, he apparently never followed up on his finding of mitochondrial lactate oxidation, and his seminal findings went unrecognized even by workers in the field of mitochondrial energetics. First to report the presence of mitochondrial LDH were Nobuhisa Baba and Hari M. Sharma, who used electron microscopy (EM) to study the hearts and pectoralis muscles of rats 53 (Fig. 1 (Fig. 2). Retrospectively, based on their seminal observations of mLDH using EM, Baba and Sharma were probably the first to use the term "lactate shuttle." Regrettably, they never followed up on their original finding of mLDH, and similar to the discovery of Dianzani, the seminal findings of Baba and Sharma went unrecognized.
Beyond Dianzani 78 and those who replicated his seminal observation that mitochondria were capable of oxidizing lactate 13,51,52,54,56,79 were the efforts of those who failed in their attempts to isolate mitochondrial preparations that contained sufficient mLDH to oxidize lactate [47][48][49]80,81 . In addition, there were others who routinely found LDH in their mitochondrial preparations but regarded their findings as an artifact and took steps to block mLDH, thus permitting the measured rate of exogenous pyruvate oxidation to rise 82 . The lesson to take from history is that mLDH is necessary for lactate oxidation. When mLDH is lost in the isolation of single fibers 49 or mitochondrial vesicles 68 or if oxamate is used to block mLDH 13 , the preparation will be able to oxidize pyruvate but not lactate.
The ability of the mitochondrial reticulum to respire lactate is fundamental to the operation of lactate shuttles because lactate disposal via oxidation in the reticulum decreases the cellular [lactate], thus establishing lactate concentration gradients down which lactate fluxes. Hence, it is understandable that the rates and directions of lactate flux vary with physiological conditions (e.g., rest or exercise, early or late during exercise, endurance-trained or sedentary individuals, sea level or high altitude, carbohydrate nourished). For instance, in the postprandial state, when red skeletal muscles are glucose consumers and lactate producers 4 , and hence drivers of lactate shuttling, the heart, liver, and kidneys are consumers or recipients of lactate shuttle. In contrast, during exercise, white skeletal muscle and the integument are lactate producers and drivers of lactate shuttling, whereas highly oxidative (red) fiber types, cardiac tissue, liver, kidneys, and brain [83][84][85][86][87] are sites of net lactate disposal. In these and other forms of shuttling, lactate fluxes from high to low concentrations with the cellular respiratory (mitochondrial) apparatus, including mLDH, functioning as removal sites 3,5,88,89 . These phenomena underpin the tracer-measured flux rates observed under conditions of rest and exercise 35,90,91 .
In a previous publication, the presence of a Postprandial Lactate Shuttle was introduced. Following a carbohydrate (CHO) meal, glycolysis, and lactate production in noncontracting red skeletal muscle 92,93 raises lactate in the systemic circulation, thus providing a substrate for hepatic and renal gluconeogenesis 85,94 and an energy substrate for skeletal muscle 35,91,95 and the heart 39,95,96 , thereby forming a Postprandial Lactate Shuttle 4 .

TRACERS AND LACTATE TURNOVER DURING REST AND EXERCISE
Pioneering work in the field of lactate kinetics in exercising mammals was performed by Florent Depocas (1923Depocas ( -2004) and colleagues 69 . Using a continuous infusion of [U-14 C]lactate into dogs during rest and continuous steady-state exercise, they made several key, fundamental findings regarding lactate metabolism. These findings included (1) active lactate turnover during the resting postabsorptive condition; (2)~1/2 of lactate formed during rest is removed through oxidation; (3) the turnover rate of lactate increases during exercise compared to rest even if there is only a minor change in blood lactate concentration; (4) the fraction of lactate disposal through oxidation increases to approximately 3/4 during exercise, and (5) a minor fraction (1/10-1/4) of lactate removed is converted to glucose via the Cori Cycle during exercise. Although the fractions are subject to species and experimental variations, the essential results have been reproduced in rats 71 , rabbits 97  Messonnier made concerted efforts to describe the effect of exercise intensity and training state on whole-body and working muscle lactate oxidation and gluconeogenesis from lactate in humans. The effects of exercise intensity on whole-body lactate turnover and oxidation were initially reported by Stanley 70,91,95 and Mazzeo 73 , who showed that tracer-measured lactate turnover and oxidation scaled to the metabolic rate during exercise and that while oxidation accounted for~50% of lactate disposal in resting men, oxidation accounted for 75-80% of lactate disposal during continuous hard exercise.
Studies of lactate metabolism in working skeletal and cardiac muscle in men during exercise were reported by Stanley et al. in 1986 70,91 and Edward W. Gertz et al. in 1988 39,96 , respectively. The advantage of using tracer infusions along with simultaneous a-v difference and blood flow measurements across working muscle beds is that lactate uptake and net release, therefore, total production (= the sum of uptake and net release), can be determined simultaneously. Furthermore, another advantage of using tracer infusions along with simultaneous a-v 13 CO 2 and blood flow measurements across tissue muscle beds is that tissue metabolite oxidation can be measured. In addition, working muscle turnover and oxidation rates can be compared with wholebody rates. The results showed simultaneous lactate extraction (uptake) and release (production) and oxidation by working human leg muscles, which explains most whole-body lactate turnover and oxidation rates 91 . Importantly, due to intramuscular lactate extraction, turnover and oxidation, net chemical balance measurements underestimate true lactate production and Fig. 2 First report using agarose gel electrophoresis to separate LDH isoforms in mitochondria from rat liver and heart. LDH isoenzyme patterns differ between the cytosol and mitochondria in both tissues. Note cytosolic and mitochondrial LDH isoform differences between the cytosol and mitochondria across tissues. From Brooks et al. 13 .
oxidation rates for metabolites such as lactate and free fatty acids that turnover within tissue beds 102 . Subsequently, using variations of the same methodology, cerebral glucose, and lactate uptake and release were determined in traumatic brain injury patients and healthy controls 103,104 .
Another relevant but underappreciated and often unmentioned aspect of the issue of blood lactate accumulation during exercise is the assumption that the circulating lactate level rises because of net release from active muscle. Surprisingly, while net lactate release from resting muscle is common, net release from working muscle is usually transient if the power output is submaximal and held constant. As shown first by Wendell Stainsby and Hugh Welch in 1966-1967 105,106 using dog muscle preparations contracting in situ, this "Stainsby Effect" of transient muscle net lactate release at exercise onset followed by a switch to net uptake from the blood by working muscle has been confirmed in exercising humans 35 . In this regard, noteworthy are the studies of L. Bruce Gladden on dog muscles contracting in situ 107 . Gladden clearly showed that lactate uptake is the concentration (substrate) and not O 2 -dependent, a finding that also appears to be the case in human muscle 35 , vide infra. Thus, it is certain that working skeletal muscle is not the sole source of blood lactate in humans during whole-body exercise. Epinephrine is more likely to signal glycolysis and lactate production in noncontracting tissues than in working muscle; in working muscle, epinephrine augments glycolysis, leading to increased lactate accumulation [108][109][110] .
Choice of lactate carbon tracers to determine lactate flux and disposal in vivo As noted above, initial studies of lactate metabolism in vivo utilized a uniformly labeled tracer, i.e., U-14 C]lactate 69 . However, with the advent of stable, nonradioactive 13 C-labeled tracers, it was ethically feasible to study flux and oxidation in human subjects 111 . Because lactate disposal via oxidation gave rise to 13 CO 2 , carbon-labeled lactate tracers were preferred over heavy hydrogen (i.e., deuterated, D) tracers because the oxidation product ( 13 CO 2 ) could be detected at the tissue level in the venous effluent of tissues such as muscle 35 , heart 36,39 , and brain 103 and at the whole-body level by excretion in expired air 35,73,95,112 . Another advantage of the 13 C-lactate tracer was that it could be used in combination with a deuterated glucose tracer, e.g., [6,6-2 H] glucose, a so-called "irreversible tracer", because the D atoms are lost to body water during glycolysis. Moreover, because a percentage (≈25%) of 13 C atoms from lactate are reincorporated into glucose during the process of gluconeogenesis, the combination of [3-13 C]lactate and [6,6-2 H]glucose (i.e., D2-glucose) could be used to simultaneously study lactate and glucose flux rates, the lactate oxidation rate and the rate of gluconeogenesis from lactate [113][114][115] .
On the issue of which isotope tracer of lactate to use, the most common is [3-13 C]lactate, which yields information about total lactate disposal as well as the components of oxidation and gluconeogenesis. Earlier use of uniformly labeled compounds to determine metabolite disposal rates was abandoned because of the extensive recycling and exchange of labeled lactate carbon atoms 116,117 . Of the alternatives, 13 C from [1-13 C]lactate is lost as 13 CO 2 in the pyruvate dehydrogenase reaction, as pyruvate is converted to acetyl-CoA upon entry into the TCA (Krebs) Cycle. This decarboxylation step shows entry into the TCA cycle, but perhaps not complete oxidation of the lactate molecule. For that, [3-13 C]lactate is the preferred tracer to demonstrate lactate oxidation downstream in the TCA cycle by the actions of isocitrate and alpha-ketoglutaric dehydrogenases 41,118 .
Finally, with the use of carbon-labeled tracers to determine the oxidative disposal of lactate, it needs to be acknowledged that because metabolically produced 13 CO 2 enters the body's bicarbonate-CO 2 pool, a separate experiment under identical conditions needs to be conducted. At rest, the bicarbonate-CO 2 and MCT2 lactate transporter isoforms and the mitochondrial reticulum (cytochrome oxidase, COx) in adult rat plantaris muscle determined using confocal laser scanning microscopy (CLSM) and fluorescent probes for the respective proteins; comparisons for MCT1 in the first row (Plates A1-A3, respectively) and for MCT2 in the second row (Plates B1-B3, respectively). The localization of COx was detected in rat plantaris muscle (Plates A1 and B1). MCT1 was detected throughout the cells, including the subsarcolemmal (arrowheads) and interfibrillar (arrows) domains (Plate A2). MCT1 abundance was greatest in oxidative fibers where COx was abundant and the signal was strong. When signals from probes for MCT1 (green) and COx (red) were merged, the superimposition of the two probes was clear (yellow), a finding prominent at the interfibrillar (arrows) and sarcolemmal (arrowheads) cell domains (Plate A3). In contrast, the signal for MCT2 (Plate B2) was weak and relatively more noticeable in fibers denoted by strong staining for COx (Plates B1 and B3, broken line delineated around oxidative fibers to distinguish the faint signal for MCT2). The overlap of MCT2 and COx is insignificant, denoted by the absence of yellow in Plate B3. Scale bar = 50 μm. Sections are from the same animal. Reprinted from Hashimoto et al. 154 .
pool turns over slowly, so for the short term (1 to several hours), an isotope dilution factor needs to be applied 119 . In resting humans, the correction factor for CO 2 retention is large (≈50%) 120 . The large correction factor for estimating isotopic dilution and retention is daunting for investigators to make because it is so large. Fortunately, however, during exercise, the necessity of a bicarbonate correction factor disappears because the bicarbonate-CO 2 pool turns over rapidly with no measurable 13 CO 2 retention or diversion of tracer to other, unmeasured metabolite pools 121,122 . In addition, some investigators have explored the use of so-called "acetate correction factors" 123 . However, the latter approach is not recommended because acetate is not bicarbonate and, most importantly, because the C1 and C2 of acetate behave differently, leaving an investigator uncertain about what the appropriate correction factor is 120 .

THE LACTATE SHUTTLE AND MEMBRANE LACTATE TRANSPORT AND LACTATE/PYRUVATE EXCHANGE
With knowledge of glucose and lactate fluxes and oxidation rates in resting and exercising rats 71,124 , in 1984, George A. Brooks took a unique approach to explain phenomena related to lactate responses to exercise when the "Lactate Shuttle Hypothesis" was articulated 125 . A key element of the hypothesis was that "the shuttling of lactate through the interstitium and vasculature provides a significant carbon source for oxidation and gluconeogenesis during rest and exercise". As such, the lactate shuttle hypothesis represents a model of how the formation and distribution of lactate represents a central means by which the coordination of intermediary metabolism in diverse tissues and different cells within tissues can be accomplished. The initial hypothesis was developed from the results of original isotope tracer studies conducted on laboratory rats in the Brooks Laboratory along with numerous other studies, many of which were cited in the previous section. Thus, the working hypothesis was developed that much of the glycolytic flux during exercise passed through lactate.
According to the Cell-Cell Lactate Shuttle hypothesis, lactate is a metabolic intermediate rather than an end product 3,88,[126][127][128][129] . Lactate is continuously formed in and released from diverse tissues, such as skeletal muscle, skin, and erythrocytes, and also serves as an energy source in highly oxidative tissues, such as the heart, and is a gluconeogenic precursor for the liver and kidneys. Lactate exchanges among these tissues appear to occur under various conditions, ranging from postprandial rest to sustained postabsorptive exercise 88,89,125 .
If lactate does serve as a key metabolic intermediate that shuttles into and out of tissues at high rates, particularly during exercise, then transmembrane movement is critical. For many years, lactate was assumed to move across membranes by simple diffusion. However, by 1980, facilitated protein carrier-mediated transport of lactate across erythrocyte membranes was well documented 130,131 . It was not until 1990 that the characteristics of sarcolemmal membrane lactate transport were described [132][133][134] . The study of cell membrane lactate transport proteins took another major leap in 1994. When looking for the mevalonate (Mev) transporter gene in Chinese hamster ovary cells, Christine Kim Garcia in the Goldstein and Brown laboratories cloned and sequenced a monocarboxylate transporter that she and her colleagues termed the monocarboxylate transporter (MCT) 135 . In 1985, Goldstein and Brown shared the Nobel Prize for "their discoveries concerning the regulation of cholesterol metabolism" 136 . The newly discovered MCT was abundant in erythrocytes, the heart, and the basolateral intestinal epithelium. MCT was also detectable in oxidative muscle fiber types but not in the liver. With an interest in describing a role for MCT isoforms in the Cori Cycle, Garcia et al. 137 subsequently described the isolation of a second isoform (MCT2, in effect renaming the first transporter discovered to MCT1) by screening a Syrian hamster liver library; MCT2 was initially found in the liver and testes but subsequently also in the brain 138,139 and some tumor cell lines 16,140,141 .
Independent of Garcia et al. 135,137 , Price et al. 142,143 identified another MCT isoform (now known as MCT4) in 1998. In terms of physiology, Halestrap and colleagues 144 continued to hold traditional, Hill-Meyerhof views of an association between lactate transporter expression and oxidative metabolism even though their data showed a high correlation between MCT1 expression and mitochondrial markers 144,145 , which was subsequently replicated by Dubouchaud et al. 15 . However, with a different concept based on the knowledge that lactate was formed and oxidized continuously within muscle and heart tissue in vivo, Brooks and associates sought to identify the molecular basis of the coupling of glycolytic with oxidative pathways. In that pursuit, Brooks and colleagues first observed the presence of LDH in rat liver, cardiac and skeletal muscle mitochondria 15 . Subsequently, they also showed MCT1 to be in mitochondria of the same tissues 50 , thus allowing mitochondria to directly oxidize lactate 13 . With such knowledge, in 1998, Brooks extended the Cell-Cell Lactate Shuttle concept to include an intracellular component, the "Intracellular Lactate Shuttle." As already noted, several of the observations, such as the presence of mLDH 53 and mitochondrial lactate oxidation 55 , were similar to the vision of Polyphemus-without depth perception, swamped by the mediocrity of authority, buried and forgotten in the literature for a time but later resurrected 3,4 .

COMPARTMENTATION ISSUES AND CONTROVERSIES-THE INTRACELLULAR LACTATE SHUTTLE
While there is growing agreement on some aspects of the Cell-Cell Lactate Shuttle, within exercise physiology and other fields, such as neurobiology 139,[146][147][148][149][150] and cancer research 140,141,151,152 , some aspects of the Intracellular Lactate Shuttle remain controversial. In essence, disagreement exists over where the first step in lactate oxidation (i.e., conversion to pyruvate) occurs. This is why a discussion over whether mitochondria contain LDH persists. If so, identifying where LDH would reside so that it is able to oxidize lactate (vide supra) is important. However, the results of light and electron microscopy studies conducted to date show that mLDH resides within the mitochondrial reticulum.

Electron microscopy
Prior to the MitoCarta and MitoMiner databases based on proteomics, those who looked for muscle mLDH located it in tissues. Studying rat heart, Baba and Sharma used methods to protect mLDH during tissue fixation for electron microscopy. Reaction products of mLDH seen in the mitochondria were "primarily located within the cristae" (Fig. 1) (their Fig. 11) 53 . In addition, they reported similar results for rat pectoralis Types I and II fibers. Similarly, using an antibody to LDH and gold particle secondary labeling, Brooks et al. also showed LDH located on the inner mitochondrial membranes of rat muscle, heart, and liver 13 . Moreover, they used agarose gel electrophoresis to separate LDH isoforms in the cytosol and mitochondria isolated from rat livers and hearts. LDH isoenzyme patterns differed between the cytosol and mitochondria in both tissues (Fig. 2).

Immunocytochemistry
In the Brooks Laboratory, Takeshi Hashimoto used confocal laser scanning microscopy, immunoprecipitation (IP), and cell fractionation techniques followed by western blotting to verify the presence of the purported Mitochondrial Lactate Oxidation Complex (mLOC) in cultured myocytes as well as in thin sections of adult rat tissues. The colocalization of MCT1, mitochondrial cytochrome oxidase (COx) and LDH in the mitochondria of adult rat skeletal muscle and astrocytes was visualized by means of combinations of primary and fluorescently labeled secondary antibodies plus MitoTracker Red and dual-wavelength scanning confocal microscopy (Fig. 3) 138,153,154 . Subsequently, similar techniques allowed the investigators to show the colocalization of mLOC components in cultured L6 (rat muscle-derived) cells (Fig. 4). Again, those results were confirmed by two independent techniques-IP and Western blotting of isolated cell fractions 138,[153][154][155] . The results of IP efforts are shown in Fig. 5, and a pictorial representation of how the mLOC is organized on the inner mitochondrial membrane with projection into the intermembrane space is constructed from the results of immunocytochemistry and immunoprecipitation studies (Fig. 6).
Both morphometry and the susceptibility to loss during mitochondrial isolation suggest that LDH resides in the intermembrane space or, similar to cytochrome c, is loosely fixed to the inner membrane. On the other hand, if it is acknowledged that cardiac and red skeletal muscle take up and oxidize lactate, while the presence of mLDH and an mLOC are denied, then opponents of the Intracellular Lactate Oxidation Complex must rely on cytosolic oxidation of lactate to pyruvate. Cytosolic oxidation of Fig. 5 Results of efforts to deduce the organization of the mLOC using immunoprecipitation (IP) technology. In the upper panel, representative immunoblots (IB) are shown using anti-COX, NADHdh, LDH, or nIgG as precipitating antibodies (IPs). COX, NADH-DH, LDH, and nIgG were immunoprecipitated from mitochondrial fractions of L6 cells resuspended in a suspension medium without detergent. COX IP proteins were probed with MCT1, CD147, and LDH antibodies. MCT1, CD147, and LDH were coprecipitated with COX. NADH-dh IP pellets were probed with MCT1, COX, CD147, and LDH antibodies. Neither MCT1, CD147, nor LDH coprecipitated with NADH-DH, whereas COX coprecipitated with anti-NADH-dh. LDH IP proteins were probed with MCT1 and COX antibodies. Both MCT1 and COX coprecipitated with LDH. No protein coprecipitated with nIgG from mitochondrial fractions of L6 cells resuspended in medium without detergent (negative control). In the lower panel, the degree of coprecipitation evaluated by comparing signals in the IP and the lysate is shown. The results were categorized into four levels: +++, 80% or more precipitated; ++, ∼50% precipitated; +, 20% or less precipitated; -, no precipitation. IB immunoblot, IP immunoprecipitation. From Hashimoto et al. 138 .
lactate is unlikely 156 and contrary to the evidence available from Peter Hochachka and colleagues, who studied glycolysis in a variety of mammalian species 157,158 , and Bradley Zinker et al. 159 in the David Wasserman laboratory, who studied glucose and lactate metabolism in the hind limb muscles of running dogs. By means of tracer glucose, they showed intramuscular lactate production from glucose to occur simultaneously with net muscle lactate uptake 159 . Clearly, glycolysis progressed to lactate production in the working muscles of running dogs, whereas lactate oxidation occurred in a compartment where it was oxidized, the mitochondrial reticulum.
Mitochondrial monocarboxylate uptake mMCTs, mPCs or Both? Analogous to the controversial history of mLDH discovery was the discovery of the presence of mitochondrial monocarboxylate transporters (mMCTs) 13,50,68 . These were visualized in adult rat skeletal muscle 154 and cultured L6 myocytes by Takeshi Hashimoto and colleagues 153 , Figs. 3 and 4, respectively. For a time, the isoform monocarboxylate transporter 1 (i.e., MCT1) was the only lactate/pyruvate transporter known to be expressed in the mammalian mitochondrial proteome 57 . Subsequently, the mitochondrial pyruvate transporter (mPC) was identified 160,161 . Unfortunately, at present, little effort has been expended to evaluate functional or morphometric relationships between mPCs and mMCTs, particularly in mammalian skeletal muscle. Indeed, we cannot find reports in the literature of an mPC in mammalian or human skeletal muscle. Reports of an mPC in yeast 160,161 are unconvincing because of single-band Western blots that are unaccompanied by a ladder to assess the molecular weights of the putative mPC. However, with access to our own custom antibodies to MCT1, as well as a commercially available antibody to the putative mPC, we obtained images assessing the colocalization of MCT1 and mPCs in L6 cells. In those preliminary studies, colocalization analysis of mMCT1 and mPC1 in Imaris software showed an r 2 of 0.8. It appears that both MCT1 and mPCs are colocalized to the mitochondria (r 2 = 0.8) (Fig. 7) 6 . However, at the light microscopic level, it is impossible to know whether mMCTs and mPCs interact physically and functionally. Immunoprecipitation, X-ray crystallography, mass spectrometry, and protein deletion (knockout) studies are needed to definitively answer questions about mMCT and mPC colocalization and functionality and the role of mPCs in mitochondrial lactate oxidation.
Role of the mitochondrial lactate oxidation complex (mLOC) in lactate/pyruvate metabolism Discovery of the mLOC (Fig. 5) and its role in Cell-Cell and Intracellular lactate shuttles may cause some traditional thinkers to assert something such as "the diversion of glycolytic flux from pyruvate to lactate via cytosolic LDH (cLDH) with a return to pyruvate via mLDH still means that pyruvate is the major mitochondrial energy substrate". Such an assertion would hold at the unicellular organismic level. However, to briefly reiterate from above, lactate shuttles are based on lactate concentration differences between producer and recipient cells connected through the interstitium within a tissue bed or the vasculature at the organ system level. In the shuttling of carbohydrate carbon energy substrates, lactate concentrations are orders of magnitude greater than those of pyruvate and alanine. Hence, corporal energy substrate distribution is accomplished with lactate, not pyruvate, as the vehicle of mass-energy transfer 60 .
The mitochondrial lactate oxidation complex (mLOC) in cancer As noted above, in the Brooks laboratory, Takeshi Hashimoto et al. showed the presence of the mLOC in mammalian skeletal muscle, muscle cell lines, neurons, and primary neuronal cell cultures 138,153,154 . In addition, his work revealed the presence of a lactate-sensitive transcription network in cultured myocytes 155 . Having been part of the team previously working on muscle and brain, both postmitotic tissues, Rajaa Hussien in the Brooks laboratory commenced work on breast cancer cells 140 that display active mitosis. Initial efforts were on known mLOC proteins, including monocarboxylate transporters (MCT1, MCT2, and MCT4), a scaffold glycoprotein (CD147), lactate dehydrogenase (LDH isoforms A and B), and cytochrome oxidase (COx). The results were consistent with their primary hypothesis that mLOC expression and assembly are dysregulated in breast cancer 140   proteins (MCT2, LDH and CD147) were upregulated. Remarkably, the expression of MCT1 was low in MCF-7 cells and undetectable in MDA-MB-231 cells. A notable novel discovery from their study was the reciprocal relationship between the MCT1 and CD147 protein levels. The data from breast cancer cell lines led to the hypothesis (as yet incompletely explored) that the dysregulation of the coordinated expression of those two proteins and their respective genes contributes to the carcinogenic process in breast cancer.
To probe for the presence of mLOC proteins in cancer cells, Hussien and Brooks 140 also examined the subcellular localization of MCTs and LDH isoforms in the three cell lines studied. Confocal laser scanning microscopy and related immunohistochemical techniques showed the presence of MCTs, LDH isoforms, and COx. LDH isoforms MCT2 and MCT4 were colocalized with the mitochondrial protein marker COx, but distinct from the situation in muscle, MCT1 was not mitochondrial and was localized exclusively in the plasma membrane. The localization of MCT and LDH isoforms in the two cancer cell lines (MCF-7 and MDA-MB-231) and the normal cell line (HMEC-184) was the same. MCT2, MCT4, and LDH were localized in mitochondria in addition to their localization in the plasma membrane and cytosol, whereas MCT1 was mainly localized in the plasma membrane (Fig. 8). Their data showed that in a breast cancer cell line, changes in the expression of lactate shuttle proteins occur. The reasons for these changes in mLOC protein expression, transcription, translation, and turnover in breast cancer need elucidation.

LACTATE SIGNALING
The effects of increases in cellular work and subsequent lactate production on redox, reactive oxygen species (ROS), allosteric binding, and histone lactylation have been recently reviewed 5 . Briefly, lactate affects metabolic signaling via diverse mechanisms that include changing the L/P and NADH/NAD + and thus cell redox; activating sirtuins via the enzyme NAM phosphoribosyl transferase (Nampt); stimulating transforming growth factor beta 2 (TGF-β2) secretion from adipose tissue; regulating gene expression by the lactylation of 28 lysine residues on histones 162 ; and inhibiting lipolysis by activating Hydroxycarboxylic Acid Receptor-1 (HCAR-1) operating through cyclic AMP (cAMP) and cAMP response element-binding (CREB 163,164 . With regard to gene regulation via histone lactylation, it should be noted that it was discovered using tracer methodology ([U-13 C]lactate and [U-13 C] glucose) and a cancer cell line (MCF-7). As noted previously, the purported effects of lactate signaling via HCAR-1, TGF-β2, Nampt, and the lactylation of histones observed in rodent muscle and brain and cell line models await validation in humans 4 . The latter statement is appropriate because of the growing interest in evaluating the role of lactylation in promoting carcinogenesis in a variety of cell types, including those of the lungs 165 . Regrettably, thinking in the field is limited by reliance on outmoded O 2 debt (i.e., O 2 -limited) ideas of metabolic regulation 166 .

LACTATE AND MITOCHONDRIAL BIOGENESIS
Endurance exercise has long been recognized to stimulate mitochondrial biogenesis 167,168 . Hence, the following question arose: 'Could the lactatemia of exercise stimulate mitochondrial biogenesis?' In an attempt to address the question, Hashimoto and colleagues incubated L6 cells in elevated sodium lactate added to buffered, high-glucose media and screened the cells' genome-wide responses 155 . Lactate increased ROS production and upregulated 673 genes, many known to be responsive to exercise and exercise training via ROS and Ca ++ stimulation. The induction of genes encoding components of the mLOC was confirmed by a polymerase chain reaction and electrophoretic mobility shift assay (PCR and EMSA, respectively). Among the many effects, lactate increased the mRNA and protein expression of MCT1 and COx. Increases in COx expression coincided with increases in peroxisome proliferator activated-receptor gamma coactivator-1 alpha (PGC1α) expression and the DNA binding activity of nuclear respiratory factor (NRF)-2. Based upon their results, Hashimoto et al. concluded that the presence of a lactate signaling cascade involves ROS production and converges on transcription factors affecting mitochondrial biogenesis.
The presence of a lactate-sensitive transcription factor network and its putative effects on muscle mitochondrial biogenesis in exercising humans have yet to be evaluated. To date, Genders et al. 169   lactate on histone deacetylase (HDAC) activity and protein kinase B (aka, Ak strain transforming, Akt) signaling in L6 cells. Studies on the possibility of exercise above the lactate threshold, where there is a disproportionate increase in muscle lactate production, activating a signaling cascade leading to human skeletal muscle mitochondrial biogenesis are underway (David Bishop personal communication).

THE LACTATE STORY COMES FULL CIRCLE
Considering the above text on intact individuals in vivo, tissues, and organs in situ, and cells in vitro, as well as the results on cancer cells to date, it seems that the study of lactate metabolism has come full circle. From the perception of a lowly waste product formed as the result of a lack of oxygen, as embodied in the shuttle hypothesis, lactate is now realized to have three essential metabolic functions: an energy source, a gluconeogenic precursor, and a signaling molecule. Originally conceived of within the context of exercise physiology and supporting short-and longterm adaptive processes, some investigators propose using lactate esters and salts to sustain athletes in prolonged hard exercise bouts 170 and to provide nutritive support to humans following traumatic brain injury 104 .
Regrettably, in clinical settings, the understanding and use of lactate in diagnosis and treatment is inadequately understood and employed. For instance, half molar lactate infusion is useful to supply nutritive support to patients with heart failure and following myocardial infarction 171 . Similarly, in the realm of traumatic brain injury 104 , a clinical trial is underway to evaluate the efficacy of lactate supplementation to support the injured brain. Nevertheless, as lamented by See and Bellomo 172 , in intensive care units where the blood lactate level is a biomarker for the severity of sepsis, a lack of knowledge on the sites and reasons for lactatemia hampers treatment. Fortunately, progress has been made in understanding the role of lactatemia in the etiology of diabetic ketoacidosis 173 . Similarly, the role of dysregulated lactate metabolism is advancing, with investigators giving thought to destroy cancer cells by blocking lactate shuttling 140,141,151,174,175 .

SUMMARY
A chronology of significant events and important discoveries in the field of lactate metabolism is given in Table 1. As shown, in the latter part of the 20th century, the use of isotope tracer technologies enabled significant contributions to a revolution in understanding the diverse roles of lactate in the regulation and integration of intermediary metabolism in humans and other mammals in health and disease.
Isotope tracer technologies were central to the deduction and demonstration of the Lactate Shuttle at the whole-body level. In concert with the ability to perform tissue metabolite concentration measurements, as well as determinations of unidirectional and net metabolite exchanges by means of a-v difference and blood flow measurements across tissue beds including muscle and the brain, lactate shuttling within organs and tissues was

1907-1924:
The Lactic Acid Era. Archibald Vivian (A.V.) Hill and the Cambridge School of Physiologists believed that the "processes of muscle contraction are due to the liberation of lactic acid from some precursor" [22][23][24]176 . Otto Meyerhof quantifies the relationship between glycogen and lactic acid formation in isolated, nonperfused frog hemicorpus preparations [25][26][27] . A.V. Hill develops the O 2 Debt concept in human studies [65][66][67]177 . August Krogh and Johannes Lindhard 178 provide evidence supporting Zuntz's assertion that both fat and carbohydrate are substrates for energy during exercise in humans 179 , but those studies were outside the prevailing theory of the Cambridge School of A.V. Hill and associates.
1925: Contrary to the oxygen-limited, O 2 Debt Ideas of the Cambridge school, contemporaneously in Germany, Otto Warburg describes aerobic glycolysis in tumor cells; subsequently the phenomenon became known as the "Warburg Effect" 64,180 . Later, it became obvious that lactate production occurs in the soma of healthy individuals while resting or exercising or after carbohydrate nutrition 181 as well as in the gut microbiome 3 .
1929: Carl Ferdinand Cori and Gerty Theresa Cori propose and describe a metabolic pathway in which L-lactate produced by anaerobic glycolysis in the muscles moves to the liver where it is oxidized to pyruvate to glucose, which then returns to the muscles and is metabolized 182 . This was the first expression of a Lactate Shuttle mechanism involving the vascular exchange of precursor from a driver cell or tissue (muscle) and receipt by a recipient cell or tissue (liver), and return. 1940: After the elucidation of the glycolytic pathway (known as the Embden-Meyerhof-Parnas Pathway) that assumed pyruvate to be its end product under aerobic conditions, together with conclusions of Krebs and associates that pyruvate is the molecule that enters the TCA, most of the research into energy metabolism would continue unabated using the basic paradigm established by the principal players of the first half of the 20th century.
1951: Mario Umberto Dianzani shows that rat liver mitochondria oxidize lactate 78 . 1970: Albert Lehninger publishes the textbook Biochemistry 41 . Concepts of glycolysis and fermentation are confused. Ideas that glycolysis leading to pyruvate under aerobic conditions and that lactate is produced due to oxygen lack were enshrined in the latter part of the 20th century. Only recently has a contemporary understanding of distinctions between fermentation and glycolysis, and the role of oxygen, or rather the lack thereof, in determining the end product of glycolysis which is lactate have started to emerge in contemporary basic biology textbooks 40 . 1971: Nobuhisa Baba and Hari M. Sharma visualize mitochondrial LDH using electron microscopy. They were the first to postulate a "Lactate Shuttle", but did not follow up on their observation 53 .
1973: George A. Brooks and associates give 14 C-lactate to rats after exhausting exercise and find, contrary to classic "O 2 Debt" theory, little incorporation of lactate into glycogen but major disposal as 14 CO 2 . This is the first challenge to the classic Hill-Meyerhof concept of a 1/5 -4/5 lactate-to-lactate conversion to glycogen ratio, which is converse of what happens in a mammalian system after exercise 191 .
1980: Glenn A. Gaesser and George A. Brooks use bolus injections of [U-14 C]glucose and -lactate tracers, indirect calorimetry, and two-dimensional chromatography to trace the paths of lactate and glucose disposal during recovery from exhausting exercise. Again, they find little incorporation of lactate-derived carbon into glycogen but major disposal as 14 CO 2 . In mammals, oxidation, not reconversion to glycogen, is the major fate of lactate after exercise 116,117 .
1983: Casey M. Donovan and George A. Brooks use primed continuous infusions of [U-14 C]glucose and -lactate tracers, indirect calorimetry, and two-dimensional chromatography to determine the flux, oxidation, and conversion rates of lactate to glucose in endurance-trained and untrained rats during exercise. Training results in the classic finding of lowered arterial [lactate] that is due to increased clearance via oxidation and gluconeogenesis 71,124 . This seminal study on lab rats and the resulting paper was subsequently reproduced using stable, nonradioactive tracers on human subjects 35,[112][113][114]192,193 .
1984: Richard Connett, Tom Gayeski, and Carl Honig observe lactate production in canine muscle in situ when intramuscular PO 2 is apparently above the critical value for mitochondrial oxidative phosphorylation 30 .
1984: Lactate Shuttle Hypothesis Articulated: Based on tracer-measured glucose and lactate fluxes and biochemical evidence from the Kenneth M. Baldwin Lab 194,195 , George Brooks proposes the Lactate Shuttle in a meeting of comparative physiology in Liege, Belgium. Meeting proceedings are published the following year 125 .
1984: Daniel Foster presents at the annual Banting Lecture, revealing the importance of lactate to hepatic glycogen synthesis (the 'Indirect Pathway') following carbohydrate nutrition 196 . In retrospect, Foster's work anticipated the Postprandial Lactate Shuttle 181 .
1984: George A. Brooks and Thomas D. Fahey publish the first edition of EXERCISE PHYSIOLOGY: HUMAN BIOENERGETICS AND ITS APPLICATIONS 118 . Contemporary ideas of lactate metabolism appear in a textbook. In two volumes, the work is now in its fifth edition 46,197 . Textbook versions of the Oxygen Debt and Anaerobic Threshold were fundamentally changed. 1988: For the first time, Avital Schurr and colleagues described the ability of lactate to support synaptic function of hippocampal neurons in vitro as the sole source of energy substrate 198 . evident. We now know that Organ-Organ, Cell-Cell, and Intracellular Lactate Shuttles operate continuously, even after eating when a Postprandial Lactate Shuttle is evident 4 . By means of lactate shuttling, fuel-energy substrate exchange occurs between tissues and organs such as muscle and the brain; muscle and the heart; muscle and the liver, kidneys, and gut; and muscle and other tissues. A muscle-like role of the integument is also highly suspected but unexplored. A Corporal-Microbiome Lactate Shuttle has been proposed 3 but is also unexplored. Within tissues, lactate can be exchanged between white and red fibers within a muscle bed and between astrocytes and neurons in the brain. Within cells, lactate can be exchanged between the cytosol and mitochondria and between the cytosol and peroxisomes. By means of isotope tracers and related methodologies, we know that there are at least three general functions of lactate shuttling: lactate is an energy source preferred over glucose in the heart, red skeletal muscle, and brain; lactate is the major gluconeogenic precursor; and lactate is a signaling molecule. Lactate signals otherwise affect energy substrate partitioning by mass action, cell redox, the lactylation of histones, and covalent binding to HCAR-1 and signaling via CREB-dependent pathways. Discovered in the realm of exercise physiology and biochemistry, it is now possible to recognize a Postprandial Lactate Shuttle by which dietary carbohydrates are absorbed, circulated, and taken up and used or stored in diverse tissues. Isotope tracers and related technologies have made multiple fundamental discoveries possible in the field of metabolic regulation.