State of the Art, circa 1990
In the proceedings of the 1990 Annual Meeting of the ACSM (12), the following findings were described:
1. Electrically stimulated dog muscle preparations studied in situ release lactate on a net basis when contractions start, but switch to net consumption as contractions continue (103).
2. Measurements using NADH fluorescence (59) and myoglobin cryomicroscopy (24) on electrically simulated canine muscles studied in situ indicate that lactate production occurs under fully aerobic conditions.
3. During continual, progressive exercise working human muscle is a site of net lactate production and release as well as the site of blood lactate appearance (93,94).
4. During constant rate, submaximal exercise working human muscle first releases lactate on a net basis when exercise starts but then switches to net lactate uptake as contractions continue (1,16,20,81,99).
5. Perfusion with high blood lactate causes canine muscle contracting in situ to switch from net lactate release to net consumption (45,46).
6. Addition of arm to leg cycling exercise causes arterial lactate concentration ([lactate]) to elevate. Consequently, when the arterial [lactate] rises, exercising legs switch from net lactate release to net consumption (81). Thus, in both dog muscle contracting in situ and working human muscle in vivo, elevation of arterial lactate delivery to working muscle has a similar effect on shifting the balance of substrate utilization.
7. In resting humans, glucose rate of disappearance (Rd) exceeds the rate of lactate appearance (Ra), but during exercise the gain in lactate Ra is much greater than the gain in glucose Rd (95), such that during hard exercise lactate Ra and oxidation rate (Rox) exceed that of glucose (15,16,95).
8. During exercise, epinephrine secretion is exponentially related to relative exercise intensity (65), and lactate Ra and epinephrine concentration ([epinephrine]) are highly correlated (16). However, slope of the lactate Ra-[epinephrine] relationship is much greater during exercise than during rest. These results suggest that contraction, not epinephrine, is the major stimulator of glycolysis in muscle.
9. Working healthy cardiac muscle is a net consumer of lactate, but free fatty acids are the major energy substrate for the heart in resting subjects. However, during exercise when arterial [lactate] rises, exogenous (arterial) lactate becomes the major fuel for the heart (41,42).
10. Lactate is the major gluconeogenic precursor during exercise, with gluconeogenesis contributing 25–30% to hepatic glucose production in both laboratory rats (17,30,31,97,98) and humans (95).
11. Paradoxically, lactate, not glucose is the preferred substrate for hepatic glycogen synthesis. Thus, the terms “glucose paradox” and “indirect pathway” of liver glycogen synthesis after eating were emerging and becoming accepted by the wider scientific community (6,27,35).
12. Lactate exchange through cell membranes in diverse tissues was suspected to be carrier mediated. Facilitated exchange through cell membranes of erythrocytes (29,49), cardiocytes (96), intestine (85), and by intact skeletal muscles was known (86,87). Further, Roth and Brooks (86,87) had recently demonstrated carrier-mediated lactate uptake by sarcolemmal vesicles. Lactate uptake by sarcolemmal vesicles demonstrated: concentration dependence, saturation, competitive inhibition by other monocarboxylates, inhibition by known monocarboxylate inhibitors, stereospecificity, temperature, and hydrogen ion dependence. Further, trans-stimulation was suspected and soon demonstrated (22).
As a result of the knowledge available at the time, at the 1990 Annual ACSM Meeting the lactate shuttle hypothesis was depicted as originally portrayed (Fig. 1). Features of the hypothesis included: lactate production at sites of high glycolytic flux (e.g., Type IIB muscle fibers), lactate uptake and removal at highly aerobic sites (Types I and IIA muscle fibers), lactate transport through the interstitium and vasculature with oxidation and gluconeogenesis representing major routes of lactate disposal in heart and liver, respectively.
The Current State of the Art, 1996
Since the last ACSM symposium in 1990, some significant progress has been achieved as evidenced by publication of Dr. Gladden’s reviews (43,44) and these proceedings. Further, there is evidence that the coming year will yield new and important discoveries in the areas of whole-body and tissue lactate kinetics, and the biochemistry and molecular biology of lactate exchange and metabolism. However, for the interim, it is important to record progress in three areas: the role of working muscle in net lactate release and consumption, the role of lactate as a gluconeogenic precursor during exercise, and identification of the family of cellular monocarboxylate transport proteins.
Working Muscle Net Lactate Release and Uptake
Our collaborative studies on Pikes Peak have been invaluable in allowing us to evaluate the interactive effects of ambient hypoxia and exercise on glucose and lactate metabolism (15,16,20,21,82,83). Using data from the 1988 study, I address two hypotheses regarding lactate metabolism: 1) muscle lactate formation is the result of O2 lack, and 2) working muscle is the source of elevated blood lactate in subjects during exercise and at altitude.
In 1988, and again in 1991, we studied men during continuous leg cycling exercise at a power output that elicited 50% of V̇O2max at sea level. The same subjects were studied on arrival at 4300 m (PB = 463 Torr) and after 3 wk of residency. At altitude, the same power output elicited 65% of V̇O2max, and residency did not change V̇O2max (21,104). Wolfel et al. (104) measured whole-body and working limb V̇O2 during sustained exercise at altitude and found O2 supply to be well-matched to demand. Thus, none of the changes in blood lactate response could be attributed to tissue O2 lack.
The arterial [lactate] response in each condition of sea level and altitude exercise is shown in Figure 2. Results display the typical pattern (33,80) of elevated arterial [lactate] during exercise compared with rest, an increase in [lactate] upon altitude exposure, and a decline to intermediate levels with acclimatization. Again, neither whole-body nor leg V̇O2 changed with altitude acclimatization.
The constancy of arterial [lactate] levels during exercise (Fig. 2) are to be contrasted with patterns of leg net lactate release shown in Figure 3 (16,21). When exercise starts at sea level, net lactate release from the legs (L = 2 × measured blood flow for one leg × [v-a]lactate) increases dramatically (Fig. 3 top), but after a few minutes, net lactate release declines even though arterial [lactate] is elevated and constant. Leg net lactate release is exaggerated at the start of exercise at altitude, but the same pattern as at sea level holds (Fig. 3, bottom and middle); initial lactate release upon exercise onset is followed by a decline such that after 15–20 min of exercise the working leg ceases to be a site of lactate net release even though arterial [lactate] is elevated.
In our 1988 experiments on Pikes Peak, we also infused [3-13C]lactate tracer (16). Thus, we were able to determine that lactate was undergoing very active turnover in the blood even though arterial [lactate] was constant. Further, lactate turnover rate correlated with the circulating [lactate] (Fig. 4). Therefore, we knew that the elevation in circulating lactate (Fig. 2) required a continuous input, which could not be ascribed to release from working skeletal muscle (Fig. 3). In fact, because we had measurements of [arterial-venous] concentration differences for lactate and tracer lactate, we knew that working muscle readily took up and oxidized lactate in a concentration-dependent manner (Fig. 5). Thus, working human limb muscles behaved like dog muscles contracting in situ (45,46). Moreover, the tracer lactate extracted by working muscle was quantitatively excreted as 13CO2, which was measured in the femoral venous blood and expired air (16). What remains unclear at present is the identity of the tissue, or tissues, responsible for the continuous lactate production and blood lactate appearance observed. Alpha-adrenergic stimulation of glycolysis in adipose tissue (34) and skin (61) has been observed, and it has been suggested that these tissues are possible sites of lactate production during exercise (20,21). In running dogs, the liver is a source of net lactate release during exercise (101), but in humans (54,95,99) and rats (17,30,31,97,98) the liver appears to participate in lactate clearance by accomplishing gluconeogenesis from lactate.
Gluconeogenesis During Exercise
Since the previous symposium, some progress has been made in the area of evaluating the importance of lactate as a gluconeogenic precursor during exercise. At present, the volume of data is small, but different methods have yielded similar results. Representative results are summarized in Table 1. Before discussing gluconeogenesis during exercise in more detail, a context needs to be provided.
During exercise, most (75–80%) of lactate is removed via oxidation, and 20–25% of the lactate flux is disposed of by conversion to glucose. We have obtained corresponding data on rats (17,30) and humans (15,16,21,95), and similar data have been obtained on dogs (28,56). However, because the lactate flux during exercise can be far larger than the glucose flux, conversion of lactate to glucose is sufficient to make a significant contribution to hepatic glucose production. In resting postabsorptive individuals, gluconeogenesis contributes approximately 15–30% to hepatic glucose production, and hepatic glycogenolysis the remainder.
The data in Table 1A were obtained by use of the so called “secondary labeling technique.” This approach involves infusion of carbon tracer lactate (e.g., [3-13C]lactate) and measurement of abundance of the label in circulating glucose (54). However, as has been well described by Hetenyi et al. (51) and others, isotopic dilution of 3-C precursors, such as pyruvate, in the TCA and other pools during gluconeogenesis requires application of correction (H) factors for estimating gluconeogenesis from 3-C precursors. Unfortunately, although the H factors are known to vary according to condition, they are difficult to estimate and usually require separate experiments. Therefore, a factor to correct for isotopic dilution due to carbon crossover during gluconeogenesis is generally assumed, rather than empirically determined. The data in Table 1A assume H = 1.0, which means no correction for isotopic dilution. Therefore, the listed values possibly represent underestimations of gluconeogenesis from lactate. The values are relevant, however, as they provide an appreciation of the major role of lactate as a gluconeogenic precursor.
Another approach to estimating gluconeogenesis is to employ a “dual tracer” technique. This technique involves infusion of hydrogen-tracer glucose (e.g., [6,6-2H]glucose, an “irreversible” tracer in which the labeled atoms are lost to body water during gluconeogenesis), and carbon-tracer glucose (e.g., [1-13C]glucose, a “reversible” tracer as the resulting carbon-labeled lactate, pyruvate, and alanine will recycle to glucose as the result of the Cori cycle) (26). Of the 3-C precursors, lactate is by far the most abundant and quantitatively important. The data presented are from the recent report of Friedlander et al. (36). In men, recycling accounted for 17% of glucose Ra at rest and 24% during exercise at 65% V̇O2max. Because the glucose flux doubled in the transition from rest to exercise, the actual recycling rate tripled.
With regard to estimating gluconeogenesis during exercise, a new approach of Hellerstein and Neese and associates termed mass isotopomer discrimination analysis (MIDA) (50,76) is emerging. This approach involves infusion of 13C-labeled 3-carbon gluconeogenic precursors (e.g., 13C-lactate or -glycerol) and observing the frequency of label incorporation into glucose. MIDA will yield less ambiguous results than secondary labeling or dual tracer approaches, and preliminary data (Trimmer et al., unpublished data) are consistent with the data in Table 1.
Sarcolemmal Monocarboxylate (Lactate) Transporters
Implicit in the lactate shuttle hypothesis of 1984 (Fig. 1) is facilitated exchange of lactate between lactate producing and consuming cells and tissues. The idea that lactate would exchange between red and white muscle fibers through the interstitium and vasculature was prompted by observations, such as those of Baldwin et al. (4), who observed that lactate content in red regions of rat muscle was lower than in adjacent white regions of the same muscle, or even arterial blood. The idea of facilitated lactate exchange along concentration gradients in human skeletal muscle was supported by observations of Jorfeldt (62), and subsequently Stanley et al. (94), that muscle lactate uptake displayed saturation. At that time, there existed no definitive data on carrier-mediated sarcolemmal lactate exchange, but the literature contained significant data that indicated presence of carrier-mediated monocarboxylate exchange in erythrocytes. Results of two groups, Halestrap and Denton (49) and Deuticke and associates (29) were prominent in those discoveries. Moreover, lactate uptake by liver (75,84) and heart (96) preparations was noted. In addition to other characteristics of carrier-mediated transport, inhibition by α-cyano-4-hydroxycinnamate (CIN) was prominent. As well, Halestrap and Denton (48,49) and Paradies and Papa (77) showed that mitochondrial pyruvate uptake was sensitive to CIN.
With regard to lactate exchange in skeletal muscle, in 1975 Mainwood and Worsley-Brown (71) showed that pH could influence lactate release from frog muscle. Subsequently, in 1988, independently Juel (63) and Watt et al. (102) working with mouse and rat muscle preparations, respectively, provided convincing evidence of facilitated and proton-linked lactate transport into muscle tissue. Those observations were followed by the work of Roth and Brooks (86,87) on isolated sarcolemmal vesicles.
Understanding tissue metabolite exchange is difficult because the factors determining metabolite uptake and release are arranged in series and parallel. Difficulties in understanding metabolite exchange by cells and tissues has resulted in reductionist approaches including the isolation of cell membranes and their reconstitution as vesicles. Thus, preparation of sarcolemmal vesicles has become a standard means to study the ability of metabolites to enter and leave muscle cells, with investigators in the field of glucose transport leading the way (47).
Utilizing the procedures of Grimditch et al. (47), Roth and Brooks (86,87) were the first to utilize sarcolemmal vesicles to characterize membrane-mediated lactate transport. Roth and Brooks (86,87) found sarcolemmal lactate transport to display characteristics of: concentration dependence, saturation and stereospecificity (Fig. 6), partial inhibition by other monocarboxylates, blockade by known inhibitors of monocarboxylate transport (Fig. 7), temperature sensitivity, and stimulation by hydrogen ion gradients. Important for understanding the physiology of tissue lactate exchange in vivo was the demonstration of high Vmax and Km values for sarcolemmal lactate transport. Those results mean that cell lactate uptake and release are responsive to gradients in lactate concentration and hydrogen ion concentration through the physiological range.
Subsequently, trans-stimulation (-acceleration) of lactate transport by sarcolemmal vesicles was demonstrated by Brown and Brooks (22). The latter result is significant as it indicates that the transporter is bi-directional, that is, transporters can facilitate lactate flux along concentration and proton gradients, either into or out of muscle cells. Importantly, results of Brooks and associates were soon replicated by others on rodent (73) and human tissue preparations (64).
The field of study of cell membrane lactate transport proteins received a tremendous boost when, in extending their earlier work on the mevalonate (Mev) transporter (66) with Chinese hamster ovary (CHO) cells, Garcia et al. (39) cloned and sequenced a monocarboxylate transporter, which they termed MCT1. MCT1 differed from Mev by only one amino acid substitution (Cys for Phe), and the amino acid structures of MCT1 and MEV predicted a membrane-bound protein with 12 membrane-spanning regions. Transfection of a breast cancer cell line lacking MCT1 with a plasmid containing cDNA encoding for MCT1 conferred properties reported for the erythrocyte transporter including increased pyruvate uptake, proton symport, trans-stimulation, partial inhibition by other monocarboxylates (including lactate), and sensitivity to CIN. With an interest to describe a role for MCT isoforms in the Cori cycle, Garcia et al. (38,40) subsequently described isolation of a second isoform (MCT2) by screening of a Syrian hamster liver library; MCT2 was found in liver and testes.
Independent of Garcia et al., Poole and Halestrap (79) and Jackson et al. (57,58) cloned and sequenced MCT1 and MCT2 isoforms from rabbit and rat tissues, respectively. Subsequently, the same group succeeded in identifying RNAs encoding for four more putative MCTs found in diverse mammalian tissues. Those results are summarized in the accompanying paper by Bonen, who presents a detailed discussion of MCT isoforms.
Notwithstanding that little is published on regulation of MCT expression in diverse tissues, it is probable that expression can be affected by chronic activity level. For example, with studies on sarcolemmal vesicles isolated from rat hind limb skeletal muscles of tail-suspended and control rats, Dubouchaud et al. (32) observed inactivity to decrease lactate transport activity. Conversely, working with an antibody to MCT1, McCullagh et al. (72) observed that chronic electrical stimulation increased MCT1 content in red and white rat skeletal muscles.
Although the level of physical activity can affect long-term expression of sarcolemmal lactate transporters (32,72), they are apparently not translocated as the result of acute bouts of muscle contraction (31a). Further, there are no known phosphorylation sites on MCT isoforms, and, other than H+, there are no known allosteric binding agents to MCT isoforms. Thus, rates of lactate flux, which can change 100-fold in working mammalian muscle, are not likely to be regulated over the short term by translocation or synthesis of cell membrane MCT proteins. Consistent with predictions of the classic cell-cell lactate shuttle (Fig. 1), tracer studies of glucose (36) and lactate (16) fluxes in resting and exercising humans show that the most lactate is removed through oxidation in skeletal and cardiac muscle cells with high mitochondrial densities. Further, much of the remainder is removed through gluconeogenesis, a process requiring mitochondrial function in the liver and kidneys. Thus, rather than by recruitment or activation of sarcolemmal lactate transport proteins (i.e., MCTs), lactate clearance is most likely controlled by mitochondrial uptake and oxidation in vivo.
Proposal for an Intracellular Lactate Shuttle
The findings that working cardiac (41,42) and highly oxidative mammalian (e.g., canine, 106) skeletal muscles simultaneously consume lactate on a net basis and convert glucose to lactate, along with numerous other observations on men and laboratory animals during steady rate exercise, are interpreted to mean that most glycolytic flux is converted to lactate and disposed of as lactate within a muscle fiber. Thus, the “lactate shuttle” hypothesis has been modified to include an intra- as well as extra-cellular component (i.e., an “intracardiocyte and myocyte lactate shuttle”) (Fig. 8) (13). If true, the hypothesis requires a reevaluation of the traditional way in which intracellular pyruvate ↔ lactate interactions are conceived to operate in vivo.
Current evidence can be interpreted to imply that in muscle both sarcolemmal (13,39,78,86) and mitochondrial membranes (13,48,77) contain monocarboxylate (lactate) transporters. With regard to the possibility of one or several mitochondrial lactate (monocarboxylate) transporters, the current literature contains the following bits of evidence. Heart and skeletal muscle homogenates (74) and isolated heart mitochondria take up and oxidize both pyruvate and lactate (8,13,13a,67). Pyruvate uptake and oxidation by mitochondria and isolated heart preparations are sensitive to inhibition by known MCT inhibitors, such as α-cyano-4-hydroxycinnamate (CIN) (7,23,45). As well, we have found that lactate uptake and oxidation by isolated mitochondria is CIN-sensitive (13a). Moreover, isolation and partial purification of a mitochondrial MCT (mMCT) has been reported for bovine heart (7). Using similar methodology, we (2) have used hydroxylapatite column chromatography to isolate and partially purify sarcolemmal lactate transport proteins. As with sarcolemmal MCT isoforms, it is unlikely that the mitochondrial form responds to allosteric activation. Rather, the mitochondrial MCT isoform likely responds to changes in substrate concentration dependent on lactate and pyruvate gradients established as the result of glycolytic production and mitochondrial consumption rates.
In addition to current evidence supporting presence of mitochondrial MCT isoforms, there exists solid evidence for an intra-mitochondrial pool of lactic dehydrogenase (mLDH). This mLDH pool, rather than cytosolic LDH, is essential in mitochondrial lactate uptake and oxidation. Depending on the tissue, this mitochondrial mLDH pool is distinct in isoenzyme pattern from that in the cytosol, and mLDH appears to consist mainly of the heart (H4, LDH-1) isoform (3,8,13,14). Location of mLDH may include the periplasmic space (8,13), or the matrix as well (3,14). Moreover, the oxidation of lactate by isolated rat heart, liver, or skeletal muscle mitochondria is oxamate sensitive, oxamate being a known inhibitor of LDH (14).
Given the presence of mMCT and mLDH, it is not surprising that isolated mitochondria consume and oxidize lactate at a rate equal to or greater than that of pyruvate (8,13a,14). Similarly, it is not surprising that data obtained with 13C-NMR spectroscopy and injection of 13C-lactate into working dog coronary circulation indicate uptake and oxidation of the tracer without labeling of the cytosolic pyruvate pool (68).
At the tissue and organism levels, the model of an “intracellular lactate shuttle” (Fig. 8) (13) makes seemingly divergent observations consistent. For instance, the model takes advantage of the finding (Fig. 6) that the Vmax and Km of sarcolemmal lactate transport are very high (86) and that trans-stimulation is a characteristic of sarcolemmal lactate transport (22). Thus, the sarcolemmal MCT population can hardly be predicted to limit or control lactate release, uptake, or oxidation under most conditions. Despite current emphasis on discovery of sarcolemmal lactate transport (MCT) isoforms (60), it needs to be remembered that lactate removal is accomplished mainly through oxidation within mitochondria of active, well-oxygenated red muscle and heart. Thus, mitochondrial respiration, not cell membrane lactate transport, reemerges as the key step in control of lactate homeostasis.
The model of an intracellular component of the lactate shuttle (Fig. 8) also predicts several other observations. Among these are that glycolysis inevitably results in lactate production because the Vmax of cytosolic LDH is the greatest of any enzyme in the glycolytic pathway and the Keq is far in the direction of product. Also, the model predicts that in a respiratory steady-state, elevation of arterial lactate concentration results in a switch from muscle lactate net release to uptake as well as suppression of glucose uptake (41,81). Similarly, the model allows that epinephrine stimulation of muscle glycogenolysis results in lactate release during constant oxygen consumption (90,91), whereas β-adrenergic blockade lowers, but does not eliminate the surge in muscle lactate release at exercise onset (20). High capacities for lactate clearance in trained men (95) and animals (30,31) result in lower muscle and blood lactate accumulations despite high production rates during exercise. Thus, the hallmark of low circulating lactate levels after endurance training is a characteristic attributable to high mitochondrial densities and lactate clearance in skeletal muscle (11,12). Therefore, increasing muscle mitochondrial density by increasing mitochondrial lactate and pyruvate transport and oxidase capacities facilitates function of the intracellular (intra-cardio/myocyte) lactate shuttle.
In the model proposed (Fig. 8), the sarcolemmal lactate transporter is shown as facilitated lactate exchange along concentration and pH gradients. At the level of the whole tissue, this arrangement is supported by observations that muscle can switch from net release to consumption (Figs. 3 and 5). As well, trans-stimulation of lactate transport into and out of sarcolemmal vesicles (22) and erythrocyte ghosts (78) supports the conclusion that sarcolemmal lactate transporters are bi-directional in their function. That the various MCT isoforms are specifically oriented trans and cis trans (in and out facing) to facilitate transport more effectively in vivo is another possibility that must be resolved.
At present, the progress in elaborating and understanding the various facets of the “lactate shuttle hypothesis” (Fig. 1) is so rapid that new results are appearing before previous results are widely appreciated. With current data, it is clear that lactate plays a key role in the distribution of carbohydrate potential energy between muscle and other cells. The role of lactate in serving as a gluconeogenic precursor dates to work of Cori (26). More recent interest has been on the role of lactate as a precursor in the synthesis of liver glycogen (35) and a fuel for muscle contraction (11,12). In general, cell-cell lactate flux appears to occur by means of facilitated exchange along pH and concentration gradients involving a family of lactate transport proteins (78,86,87), now called MCTs (38,39,105). Current emphasis is on cell membrane monocarboxylate transporters, but mitochondrial forms likely exist as well. For this reason, the “lactate shuttle” hypothesis has been modified to include a new, intracellular component involving mitochondrial lactate transporters and dehydrogenases (13) (Fig. 8). Indeed, if borne out by results of studies now in progress, the hypothesis of an intracellular lactate shuttle may likely lead to new understanding of the interrelationships among glycolytic and oxidative metabolism.
This work was supported by NIH grants DK19577 and AR42906.
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Chair: L. Bruce Gladden