We have also recently observed, in young men, that short-term exercise training (2 h·d−1, 7 d at 65% V̇O2max) increased MCT1 (8). Although it was not possible to measure lactate movement into or out of muscle directly, we did attempt to estimate this indirectly, by comparing the relationship between muscle and femoral-venous lactate during progressively increased exercise before and after training. We observed that the femoral venous lactate concentrations were increased for a given amount of muscle lactate (8). This suggests that trained muscles, in which MCT1 is increased, have an increased ability to extrude lactate. This supports our observations in chronically stimulated rat muscles, in which transmembrane lactate movement (i.e., influx and efflux) is increased when MCT1 is increased without an increase in MCT3-M/MCT4 (6,37,38).
The ability to take up lactate more readily and to extrude lactate more readily in oxidative or in trained muscles are not in conflict. An enhanced uptake capacity of lactate is particularly useful to clear lactate from the circulation after exercise. This can in fact contribute to the restitution of muscle glycogen, as increasing oxidation of lactate diverts glucose to glycogen formation (A. Bonen, unpublished data). Conversely, an increased ability to extrude lactate from the muscle cell into the blood is particularly advantageous during exercise to minimize perturbations in pHi (intracellular pH).
Two recent studies have examined the MCT isoform-specific responses to reduced and increased muscle activity. After 3 d of denervation, the concentrations of MCT1 and MCT3-M/MCT4 proteins were reduced in red and white muscles by 10–20%. This reduction was increased to ∼50% after 3 wk of denervation (Fig. 10) (64). In contrast, 7 d of chronic muscle stimulation increased MCT1 but failed to alter the content of MCT3-M/MCT4 (Fig. 10) (6). Thus, there is some evidence that the MCT isoforms may be regulated independently in skeletal muscle.
It is well known that lactate can be readily oxidized by the working heart. MCT1 has been shown to be present in the heart, where it is abundantly expressed. The heart contains 2–3 times more MCT1 than the most oxidative skeletal muscle (3,39). Recently, a number of additional other MCT transcripts have been found in human hearts (56) (Table 2).
Recently, it has been shown that the proton-coupled lactate transport system is the major route for the extrusion of H+ out of the heart, being quantitatively more important than the Na+/H+ exchanger (33,61), which is also known to assist with the extrusion of H+ (43,45). Because La− and H+ are cotransported, it would seem that the rates of extrusion of La−, and therefore H+, could be correlated with the available number of monocarboxylate carriers and/or their activity. This may be particularly important after an ischemic episode, when it is important to re-normalize the pHi as rapidly as possible to minimize injury to the heart (33).
Interestingly, kinetic studies using the fluorescent probe BCECF (2′,7′-bis(carboxyl)-5-(6)-carboxyfluorescein) sug-gest that La− transport was asymmetric in rat cardiomyocytes (62). At 23°C, the Km and Vmax of lactate efflux were three fold higher than for influx. These data could suggest that there is one monocarboxylate transporter with asymmetric transport characteristics. But, there was also a DBDS (4,4′-dibenzamidostilbene-2,2′-disulfonate)-sensitive and DBDS-insensitive transport component in the cardiac myocytes (62), which suggested that there are separate monocarboxylate transporters. Because of the three fold difference in lactate efflux compared to influx, it also seems that the different transporters may have different characteristics facilitating the efflux or influx of La− at different rates. Alternatively, a more rapid lactate efflux could also be attributable to the translocation of an MCT to enhance lactate efflux at a time of great need, such as during or after ischemic episodes. These considerations prompted us to examine a) the subcellular distribution of a recently cloned MCT transporter (MCT1) in the heart, in order to gain some insight into its physiological role, and b) to determine whether MCT1 expression could be increased with exercise training, to increase the rate of proton-coupled lactate transport.
By using high-resolution, immunogold immunocytochemistry, we were able to observe the MCT1 transporters at various sites in the heart (21) (Table 4). Interestingly, this revealed levels of MCT1 subdomains along the plasma membrane.
MCT1 was found along the plasma membrane, where myocytes appose capillaries and other myocytes (Table 4). The highest densities of MCT1 occurred in the intercalated disks (Table 4), where they avoided desmosomes and gap junctions. MCT1 appeared to be symmetrically spaced across the sarcoplasmic membrane and particles were concentrated within 50 nm of the plasmalemma. Thus, MCT1 appears to be differentially expressed along the cardiac myocyte membrane (Table 4) avoiding gap junctions and desmosomes. This demonstrates that there are separate subdomains of the plasma membrane that may differ both biochemically and functionally.
High MCT1 concentrations also occurred in T-tubules. Although most of the labeling was associated with the T-tubules, it was not possible to distinguish whether the labeling was also associated with the adjacent sarcoplasmic reticulum (which is very closely apposed to the T-tubules), due to the spatial resolution limitations of the immunogold labeling technique. Nevertheless, the most dense MCT1 labeling occurred in T-tubules that are in close proximity to mitochondria (21). This suggests that MCT1 facilitates delivery of lactate to its site of oxidation.
In a well-perfused myocardium, the presence of MCT1 at the intercalated disks, and in plasma membranes and T-tubules should ensure a high lactate transport capacity, i.e., net uptake such as during exercise. Additionally, MCT1 in apposing cardiac myocytes may facilitate transfer of lactate and protons between myocytes, particularly during ischemic episodes, when there is reduced or no diffusional exchange with blood. Of interest, also is the absence of MCT1 in endothelial cells in our studies (21), raising the question of how lactate is transferred across the capillary wall. It is likely that endothelial cells express another monocarboxylate transporter.
Notably, MCT1 was not associated with any internal membrane compartments. Therefore, our studies establish very clearly that the MCT1 transporter is unlikely to be translocated but that the functional capacity for lactate-proton cotransport is dependent on the number of available transporters. It is unknown whether the MCT1 in caveolae are a “reserve” supply that become available when necessary (e.g., during exercise when lactate is increased or during ischemic episodes when lactate and protons need to be extruded). Presently, it is thought that caveolae in cardiac myocytes occur predominantly in an open state (or recycle rapidly between open and closed states) (34), implying that MCT1 should be continually exposed to the interstitial fluid. However, given that caveolae are a known site for entry of ions and small molecules (2), the caveolae may well be a key site for MCT1 to facilitate proton-lactate cotransport.
Because exercise is well known to benefit myocardial function, it was of interest to examine whether exercise training could increase MCT1 and increase the rate of lactate movement across the cell wall. Therefore, we obtained hearts from rats in the training study above (i.e., moderate training: 21 m·min−1, 8% grade; intense training: 31 m·min−1, 15% grade) (3). In contrast to the lack of any change in skeletal muscle MCT1 with moderate training (Fig. 8), there was a marked and progressive increase in heart MCT1 during the 3-wk training period (Fig. 11). This was also accompanied by an increase in lactate uptake by the perfused rat heart (Fig. 9). When training was made more intense, MCT1 was increased again in the heart, but the increase was similar to that observed with the moderate training program (Fig. 11). However, heart lactate uptake was disproportionately greater than the increase in MCT1 with the more intense training program (Fig. 9). Whether this is an indication that more intense training increased another MCT isoform in the heart or whether this was associated with a more strategic placement of newly synthesized MCT1 along the plasma membrane, or intercalated disks or into caveolae is not known.
It may be of considerable practical interest that exercise training increased MCT1 in the heart at a lower training intensity than in skeletal muscle. Presumably, this increase in cardiac MCT1 may offer some beneficial protection against ischemic episodes by extruding protons and lactate more rapidly, thereby avoiding myocardial damage that may be caused by intracellular protons.
Our work has shown that MCT1 is expressed in muscle and heart. It appears that the physiological role of MCT1 is largely to support the oxidative disposal of lactate, because 1) MCT1 is abundantly expressed in skeletal muscle oxidative fibers and the heart, 2) there is a high correlation between MCT1 and indices of muscle oxidative metabolism and lactate uptake, and 3) the increase in MCT1 is associated with a more rapid transmembrane lactate movement. However, that is not to say that MCT1 is necessarily excluded from playing a role in the efflux of lactate from the cell. This would seem to be the role of MCT1 in Ehrlich-Lettre tumor cells, and possibly in chronically stimulated muscles in which MCT1 but not MCT3-M/MCT4 has been increased. Based on studies in the heart, it appears that MCT1 translocation does not occur (preliminary studies in muscle also support this lack of translocation mechanism, A. Bonen unpublished data). Thus, the lactate flux is dependent on the number of MCT1 proteins, and possibly their strategic location along subdomains of the plasma membrane and T-tubules.
Although MCT1 is shown to be responsive to exercise training, a sufficiently high intensity of exercise is required to observe an increase in skeletal muscle MCT1. In contrast, in the heart, MCT1 is increased at a much lower training intensity than in muscle. Along with increases in MCT1 there is a concomitant increase in lactate uptake by muscle and the heart after a period of exercise training.
The very low expression of MCT1 in white muscles suggests that it is only the few oxidative fibers in these muscles that express MCT1. However, our chronic stimulation studies indicate that given a sufficient contraction stimulus, it is possible to induce MCT1 expression in white muscle fibers without altering muscle fiber composition.
MCT2 and MCT3-M/MCT4 have been found mainly in rat liver and rat fast-twitch muscles, respectively. In the soleus muscle MCT3-M/MCT4 is much less abundant. These observations suggest that MCT3-M/MCT4 may be an important transporter for the efflux of lactate. Whether this is in addition to the efflux role postulated for MCT1 above remains to be determined. Such studies are now in progress, along with studies examining the function of MCT2 in liver (A. Bonen unpublished data).
Finally, we are obviously at the threshold of a new era in understanding the regulation of lactate movement into and out of skeletal muscle, cardiac, and liver cells. The discovery of seven MCT isoforms in the past 6 years and their tissue specific expression requires us to determine the factors that regulate their expression and their physiological role in muscle and the heart, as well as in other tissues, including liver, the brain, and tumor cells. Coexpression of several MCT proteins in the same tissue will require considerable work to examine the biological reason(s) for this level of organization. Certainly, it appears that there is tissue-specific and isoform-specific regulation of these monocarboxylate transport proteins.
Studies from A. Bonen’s laboratory have been supported by grants from the Natural Sciences and Engineering Research Council of Canada and by the Heart and Stroke Foundation of Ontario grant no. NA-3572. My group gratefully acknowledges the collaborations on the MCT work with Dr. A. P. Halestrap, Department of Biochemistry, University of Bristol, Bristol, U.K.
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Chair: L. Bruce Gladden