We thank Dr. Bruce Gladden for organizing this symposium on the lactate shuttle and meeting organizers for the opportunity to inform readers of recent work on the subject. As such, this paper is not a comprehensive review, because such are available (10,33), and other papers in the symposium extend the scope of our present effort.
Once thought to be formed as the result of oxygen lack, we now know that lactate is produced continuously under fully aerobic conditions, especially during exercise when rates of glycogenolysis and glycolysis are elevated (10,62). Also, from results of isotope tracer, arterial-venous difference mass balance, and biopsy studies (5,13), we now know that working skeletal muscle is not only the major site of lactate production, but also the major site of its removal, mainly via oxidation.
The carboxylic acids lactate and pyruvate are exchanged across lipid bilayer membranes by facilitated, proton-linked transport (63,64,70), involving a family of monocarboxylate transport (MCT) proteins (31). MCT1 is widely expressed in different tissues (34) and has been localized at plasma and mitochondrial membranes of muscle and other cells, including astrocytes and neurons (12,18,35,38,48,49,56). As part of the cell-cell lactate shuttle (CCLS) mechanism, MCT1 facilitates uptake of lactate into working human skeletal muscle from interstitium and plasma (5,27). A good example is the shuttle of lactate from working muscle to the heart, in which lactate is oxidized (32).
The seminal CCLS hypothesis (9) posits that lactate is either used within cells of formation or is exported to adjacent and anatomically distributed cells, tissues, and organs for use. Hence, lactate was thought to represent a carbon source for oxidation and gluconeogenesis as well as a vehicle for cell-cell signaling via redox changes (10,11). At present, there is good agreement on key elements of the CCLS, but there are fewer data and less general agreement on the cellular sites of lactate oxidation (33). Therefore, the main purpose of this brief review is to highlight the recent evidence that cellular mitochondrial networks are sites of lactate disposal via a lactate oxidation complex (35). A secondary purpose is further understanding of the coordination between muscle lactate production and mitochondrial biogenesis (36).
Intracellular Lactate Shuttle Hypothesis
The intracellular lactate shuttle (ILS) hypothesis (11) posits that lactate produced as the result of glycolysis and glycogenolysis in the cytosol is balanced by oxidation in mitochondria of the same cell. This mitochondrial lactate oxidation is plausible when energetics of lactate dehydrogenase (LDH) are considered. Lactate production occurs in muscle cytosol because the Keq of LDH is very high (3.6 × 104), and muscle isoforms with low KM values for pyruvate predominate (27), making lactate, and not pyruvate, the predominant end-product of glycolysis. Especially during exercise, muscle glucose uptake and glycogenolysis rise many times, causing pyruvate and lactate concentrations and the [lactate]/[pyruvate] ratio to rise (39,68). Lactate oxidation to pyruvate is unlikely in the cytosol of contracting muscle. In contrast to NAD+/NADH redox couple changes in the cytosol during muscle contractions, the mitochondrial NAD+/NADH pool becomes relatively more oxidized during exercise than at rest (41). The high [lactate], [lactate]/[pyruvate], and [H+] in cytosol are juxtaposed to low monocarboxylate and H+ concentrations in the mitochondrial matrix. These chemical and proton concentration gradients favor mitochondrial lactate influx and oxidation because the environment facilitates removal of both pyruvate (via TCA cycle) and H+ (via the mitochondrial ATPase). The presence of MCT1 and LDH in mitochondria allows mitochondrial lactate influx and oxidation. Evidence supporting, and not supporting, the ILS hypothesis is summarized in Tables 1 and 2, respectively. With regard to Table 2, Yoshida et al. (74) have recently reported on mitochondrial preparations low in LDH content and with poor lactate oxidation capability. Our view is that the harsh homogenization, protease, and Percoll gradient treatments caused loss of mLDH (18), which is now recognized to be a constituent of the mitochondrial proteome (69). According to them, for their preparations to respire lactate at a rate equivalent to pyruvate, the lactate concentration would have to be one to two orders of magnitude greater; but such is the case in vivo, according to a previous paper from the same group (68). Therefore, while in their Table 2 the authors listed their results as not supporting the ILS hypothesis (74), in our view, even with LDH-depleted mitochondrial preparations, they essentially prove the ILS hypothesis for in vivo conditions. Conceptual and technical filings of the paper by Yoshida et al. are described in our recent letter to editor of the Journal of Physiology (15), as well as on the Internet (http://ib.berkeley.edu/labs/brooks/Comnts_on_Yoshida.pdf). Also, in a soon-to-be-published report, Anna Atlante, Lidia de Bari, Antonella Bobba, Ersilia Marra, and Salvatore Passarella yet again show the presence of mLDH, this time in rat cerebellar granule cells.
;)
TABLE 1: Key evidence supporting the intracellular lactate shuttle hypothesis.
TABLE 2: Negative findings about a mitochondrial site of lactate oxidation.
CD147 (Basigin) Serves to Scaffold MCT1 to the Mitochondria Inner Membrane
Among the unanswered questions regarding the mechanism of lactate oxidation in vivo has been the identity of the mitochondrial chaperone for MCT1 and other associated lactate oxidation complex constituents. The single-span transmembrane glycoprotein CD147 (BSG, basigin) is considered to be the chaperone protein for MCT1, localizing it to the cell surface (29,44,73). However, few studies have focused on the association between CD147 and MCT1 in the mitochondrial reticulum of mammalian skeletal muscle cells. As CD147 serves to anchor MCT1 to the sarcolemma, it was reasonable to evaluate whether CD147 also served to scaffold MCT1 to the mitochondrial inner membrane, particularly in subsarcolemmal domains of the mitochondrial reticulum. Such a protein complex was posited to provide a means for lactate oxidation in the mitochondrial reticulum. Accordingly, we evaluated and visualized by confocal laser scanning microscopy (CLSM) that CD147 and MCT1 are colocalized throughout rat skeletal muscle-derived L6 cells, including the mitochondrial reticulum and sarcolemma (35). Supporting that observation of CLSM, our Western blots of cell subfractions demonstrated that CD147 was expressed in the mitochondrial fractions of L6 cells and liver (35). Additionally, immunoprecipitation of CD147 from the mitochondrial fraction of L6 cells coprecipitated MCT1 (35). Those results confirm and extend the observation of Kirk et al. (44) regarding CD147 as a sarcolemmal chaperone for MCT1. Previously, they have shown colocalization of MCT1 and CD147 at the cell surfaces of isolated rat cardiac cells by using CLSM, and, although their emphasis was on cell-surface protein expression, their micrographs also show colocalization of MCT1 and CD147 throughout transfected Hela cells, a finding not mentioned in their text.
To some, the presence of the glycoprotein CD147 in mitochondria might have been a surprise, given its previously documented function as a highly glycosylated protein in the sarcolemma. Although attempts to determine whether glycosylated proteins are present in mitochondria have been less convincing, the existence of a glycoprotein in rat liver mitochondria was previously demonstrated (20). The molecular weight of the glycoprotein approximated 45 kDa, which approximates that of CD147 (42 kDa), and that glycoprotein is a component of the inner mitochondrial membrane (20). Hence, it was not surprising to us that CLSM, Western blotting of cell subfractions, and immunoprecipitation techniques showed CD147 to colocalize with MCT1 as well as LDH and cytochrome oxidase (COX) at the mitochondrial inner membrane. However, how CD147 interacts and functions with MCT1 in plasma and mitochondrial membranes requires elucidation (44,47,72,73).
The Evidence for a Lactate Oxidation Complex at the Inner Mitochondrial Membrane
In addition to CD147, LDH was found to be associated with the mitochondrial reticulum of L6 cells by immunocytochemistry, Western blotting after cell fractionation, and CLSM (35). The interaction of these two proteins (MCT1 and CD147) and presence of LDH in mitochondria was confirmed by immunoprecipitation of mitochondrial fractions from L6 cells (35). Our findings obtained using both CLSM and immunoprecipitation indicate that the terminal mitochondrial electron-transport chain constituent COX is oriented to complex with MCT1, CD147, and LDH (35). However, we did not find an association between NADH-dh and MCT1 and LDH. The findings suggest the presence of a previously unrecognized mitochondrial lactate oxidation complex associated with complex IV at the mitochondrial inner membrane (Fig. 1). In this model, the chemical and proton gradients across the inner membrane of respiring mitochondrial networks establish the conditions necessary for mitochondrial lactate and pyruvate uptake. Further, it is suggested that the oxidizing environment of COX permits oxidation of lactate to pyruvate for subsequent oxidative catabolism of pyruvate in the TCA cycle.
FIGURE 1: Schematic showing the putative lactate oxidation complex: MCT1 is inserted into the mitochondrial inner membrane strongly interacting with its chaperone protein CD147, and it is also associated with COX as well as mitochondrial LDH (mLDH), which could be located at the outer side of the inner membrane. Lactate, which is always produced in cytosol of muscle and other tissues because of the abundance, activity, and characteristics of cytosolic LDH, is oxidized to pyruvate via the lactate oxidation complex in mitochondria of the same cell. This endergonic lactate oxidation reaction is coupled to the exergonic redox change in COX during mitochondrial electron transport. Transport of pyruvate across the inner mitochondrial membrane is facilitated by MCT1. GP, glycerol phosphate; Mal-Asp, malate-aspartate; ETC, electron-transport chain; TCA, tricarboxylic acid. Redrawn from Hashimoto et al. (
35), with permission.
By providing micrographs showing colocalization of MCT1, CD147, and COX in rat plantaris muscle (Fig. 2I), we extended the evidence for presence of a lactate oxidation complex to include mammalian skeletal muscle (16). Additionally, colocalization of COX and LDH was apparent in rat plantaris muscle (Fig. 2II). These micrographs support the existence of a lactate oxidation complex in adult mammalian skeletal muscle. Previously, Benton et al. (3) have reported that isolated rat muscle mitochondria contained MCT2; however, we (38) detected only faint signals of MCT2 located at sarcolemma of rat plantaris muscles. Hence, we favor the interpretation that MCT1 is the primary isoform for the lactate oxidation complex in muscle, but it may be that this is not universally the case for all tissues, such as in the brain (T. Hashimoto, H.-S. Cho, R. Hussien, and G.A. Brooks, unpublished data). So far, the ILS has been seen in liver, skeletal, and cardiac muscles (14,21), and brain (66). Evidently, the ILS is most active when tissue energy demands increase, such as during exercise, by which glycolysis and glycogenolysis are accelerated and intracellular and blood lactate concentrations increase. Further, in terms of muscle physiology and metabolism, the differential cellular localizations and relative abundances of the lactate oxidation complex (16,38) would contribute to the CCLS (9). Lactate formed in some muscle cells with high rates of glycolysis (e.g., type II fibers) could be readily released and transported into type I fibers, and then imported lactate could be readily taken up and oxidized by subsarcolemmal mitochondria (14,38). Also, the CCLS between neighboring cells, even in the same tissue, would be relevant in the brain in terms of the astrocyte-neuron lactate shuttle hypothesis (54). Hence, we are able to realize the centrality of the ILS by means of the mitochondrial lactate oxidation complex in the regulation of energy substrate flux and its logical imperatives, such as how to make a CCLS work in the absence of an ILS. Additionally, in combination with the well-known effect of endurance training on increasing the mass of the muscle mitochondrial reticulum as well as muscle oxidative enzymatic activities (24,25,40), the presence of the lactate oxidation complex helps explain why training increases lactate clearance via oxidation in working muscle (5). In the later part of this article, we provide the novel finding that mitochondrial constituents are physiologically coordinated by lactate.
FIGURE 2: Cellular locations of MCT1, CD147, COX, and LDH were determined using confocal laser scanning microscopy (CLSM) and fluorescent probes for the respective proteins in rat plantaris muscle, as previously described (
38).
Panel I, MCT1 was detected throughout the cells, including subsarcolemmal and interfibrillar domains of oxidative fibers (
arrows: plates a and c). CD147, chaperone protein of MCT1 (
plate b) is localized in association with MCT1 (
plate a). When these fluorescences (MCT1 (
green), CD147 (
red), and COX (
blue)) were merged, superposition of the three probes was clear (
white,
plate d).
Panel II, When the COX (
green) and LDH (
red) signals were merged, superposition of the two probes was clear (
yellow,
plate c). These micrographs indicate the existence of a mitochondrial lactate oxidation complex in rat plantaris muscle. Scale bar = 20 mm. From Brooks and Hashimoto (
16), with permission.
Regulation of MCT1 and CD147 Expression
To understand lactate metabolism of muscle cells in terms of the ILS, the precise mechanisms to upregulate the expression of MCT (especially MCT1) and their chaperones (specifically CD147) need to be elucidated. Until recently, the focus has been on the ability of exercise training and muscle contractions to increase the expression of the lactate transporter MCT1 in mammalian skeletal and cardiac muscles (2,7,17,22,27,37,57). In addition to exercise, cross innervations (4), testosterone (28), and myocardial infarction (42) have been observed to increase MCT1 expression as well. In human colon cells, butyrate, which is another substrate for MCT1, increased MCT1 mRNA and protein expression (23), and, in thyroid cells, thyroid-stimulating hormone (TSH) stimulated MCT1 expression at the transcription level (29). Among the various reports described above, it has been observed that MCT1 protein expression was rapidly upregulated in rat skeletal muscle by a single bout of exercise, but increases in MCT1 protein were not always accompanied by concomitant changes in transcript level (22). Similarly, it has been demonstrated in human colonic epithelial cells that butyrate increased MCT1 mRNA expression by the dual control of MCT1 gene transcription and stability of the MCT1 transcript (23). Results of these investigations suggest that the MCT1 protein expression might be regulated by both transcriptional and posttranscriptional mechanisms.
The precise mechanisms regulating CD147 expression, as well as its association with MCT1 expression in skeletal or cardiac muscles, have also not been fully elucidated. Jouaville et al. (43) have reported the effect of malnutrition on skeletal muscle MCT1 or CD147 mRNA expression in rats showing muscle type-specific regulation. In extensor digitorum longus muscle, malnutrition decreased MCT1 mRNA expression but increased CD147 mRNA expression. On the other hand, neither MCT1 nor CD147 mRNA was changed by malnutrition in plantaris or soleus muscle. Initial findings with CD147-null mice were that MCT1 protein levels did not track levels of mRNA expressed (55). More recent reports on CD147 KO mice show large, tissue-specific alterations in MCT1 protein expression as determined from Western blotting and immunohistochemistry (52). Also, with incubated thyroid cells, TSH increased CD147 protein levels, but CD147 transcript levels did not respond to TSH (29). These findings suggest that MCT1 and CD147 genes and proteins are differentially regulated. Our most recent findings (36) of the regulation of MCT1 and CD147 by lactate are described below.
Could It Be That Lactate Is Adaptive and Regulates MCT1 Expression and Mitochondrial Biogenesis?
In addition to serving as an oxidizable substrate and gluconeogenic precursor, is it possible that lactate also has a signaling role as a pseudohormone "lactormone" (10) in the activation of genes known to respond to acute and chronic physical activity? In preliminary studies on rat muscle-derived L6 cells to identify the physiological signals affecting lactate-transport protein expression, we noticed several things. First, the addition of Ca2+ or a reactive oxygen species (ROS) generator, such as H2O2, to the incubation medium caused rapid increases in MCT1 protein expression. Further, incubation of L6 cells in the high-glucose-containing medium typically used in tissue culture resulted in progressively rising lactate levels that were accompanied by increases in MCT1 and COX protein levels. Examination of the promoter areas for COX subunit IV and MCT1 revealed the presence of putative binding sites for transcription factors that are known readouts of ROS- and Ca2+-signaling pathways. Therefore, to explore the hypothesis that ROS generation, which may be induced by lactate, is involved in regulation of lactate oxidation complex proteins, we set about finding a means to control the culture lactate concentration levels and to determine the effects of lactate anion on gene and protein expression. We found that elevated concentrations of lactate in culture as occurs in contracting muscle in vivo is a key factor in the coordination of lactate oxidation: lactate upregulated the total mitochondrial mass and abundance of the lactate oxidation complex (MCT1, CD147, COX, and LDH) in L6 cells through ROS-signaling mechanisms (Fig. 3) (36). We also found, using GeneChip analysis, that lactate incubation upregulated hundreds of ROS-sensitive genes, suggesting the presence of a vast, lactate-activated transcription network, a lactate transcriptome. Specifically, these findings indicate that lactate stimulates ROS generation, which activates the transcription factors nuclear factor-kappaB (NF-κB), nuclear factor erythroid 2 (NF-E2, or Nrf2), nuclear respiratory factor (NRF)-2, and cAMP-response element-binding protein (CREB), leading, in turn, to increases in MCT1 gene expression. With regard to NRF-2 and CREB, their involvement in mitochondrial biogenesis (19,50,53,67) is notable. MCT1 is predominant in slow-twitch oxidative fibers (38) and is a constituent of the mitochondrial lactate oxidation complex (35). Coordination of MCT1 and mitochondrial biogenesis by NRF-2 and CREB is likely physiologically relevant for increasing oxidative lactate-clearance capacity in skeletal muscle (5,27).
FIGURE 3: Schematic diagram summarizing the effects of lactate on intracellular signaling in muscle. Contractions stimulate glycolysis and subsequent lactate production and accumulation. In combination, lactate accumulation and mitochondrial respiration induce ROS, which elicits many cell responses seen in the responses to exercise, including increased MCT1 expression, mitochondrial (mito) biogenesis, and the production of the antioxidant glutathione peroxidase (GPx). Also, lactate induces a large amount of gene expression, which is considered to be an adaptive response. With regard to mitochondrial biogenesis, the lactate-signaling pathway merges with Ca
2+ signaling as contractions increase cytosolic Ca
2+ flux. By itself, lactate increases expressions of slow-type troponin I (TnI) and myogenin, which are also know to be responsive to Ca
2+ flux via calcineurin (CaN). ROS can increase intracellular Ca
2+, which raises CaMK activity. Free Ca
2+ can also activate CaMK. In conclusion, lactate elicits a large number of adaptive responses and, thereby, would coordinate metabolism as a functional adaptation to exercise in skeletal muscle cells, such as activation of the lactate oxidation complex. From Hashimoto et al. (
36), with permission.
In the same study (36), increased CD147 and MCT1 protein contents were found in whole-muscle homogenates of L6 cells after 1 h of incubation with lactate. Similarly, lactate incubation increased both MCT1 and CD147 transcript levels within an hour. Therefore, at the tissue level, CD147 transcript and protein levels coincided.
Interactions among the insertions of scaffold (CD147) and transporter proteins (MCT1) into specific cell domains (mitochondria) are somewhat complex. In the mitochondrial fraction of L6 cells, increased MCT1 insertion was found after 1 h of incubation with lactate, but CD147 did not increase, although it was abundant in mitochondria by Western blotting. At 6 h of lactate incubation, mitochondrial MCT1 and CD147 levels did not change; however, considering the increased mitochondrial mass represented by increased COX expression in the whole-muscle homogenate of L6 cells, as well as increased gene expressions of mitochondrial import machinery TIM 13 and TIM 17 at 6 h (Fig. 3), lactate treatment upregulated the abundance of the mitochondrial MCT1 and CD147, as indicated by GeneChip analysis. Mechanisms of the coordination of TOM/TIM complexes with MCT1 and its accessory protein CD147, as well as their targeting signals to mitochondria, remain to be elucidated.
Previously, Mootha et al. (51) and Taylor et al. (69) performed proteomic surveys of mitochondria from mouse brain, heart, kidney, liver, and human heart. These investigators have provided evidence for more than 600 mitochondrial or mitochondria-associated proteins, including components of the mitochondrial lactate oxidation complex (i.e., MCT1, CD147, COX, and LDH). We (36) expanded their demonstration, and, by explaining how these mitochondrial constituents are assembled to form a lactate oxidation complex, we provided an insight on how the complex functions to facilitate lactate disposal in the L6 cells and adult mammalian skeletal muscle (38). Again, exercise training results in large increases in lactate clearance via oxidation (5) as well as in lactate oxidation complex constituents, with increases in mitochondrial biogenesis (36). Such an adaptation would facilitate lactate oxidation in skeletal muscle cells, permitting high-power outputs and glycolytic fluxes to occur while minimizing acidosis, which reveals the physiological significance of the ILS.
SUMMARY AND CONCLUSION
In summary, by using confocal laser scanning microscopy, Western blotting of cell fractions, and immunoblotting after immunoprecipitation from cell lysates, evidence has been provided (35) of strong associations among mitochondrial MCT1, CD147, LDH, and COX, but not NADH-dh. These findings advance our understanding of the mitochondrial role in cellular lactate oxidation, and they may be interpreted to indicate the presence of a terminal mitochondrial electron-transport chain component, the lactate oxidation complex, which establishes the proton and lactate concentration gradients necessary for the functioning of the CCLS and ILS. The physiological significance of the ILS is that it provides the necessary link between glycolysis and oxidative metabolism, with the product of the former being a chief substrate for the latter. Further significance of the most recent work is that in its signaling role (i.e., in its function as a lactormone), lactate production may be adaptive. During exercise, the transient elevations in tissue lactate concentration and mitochondrial O2 consumption induce ROS generation, which activates a transcriptional network signaling adaptive cell responses. Among these are increases in the lactate oxidation complex, mitochondrial biogenesis, and upregulation of antioxidant enzymes and moieties of Ca2+ signaling (Fig. 3). The results we provide here can be interpreted to mean that elevated lactate flux and concentration signals lead to adaptation of pathways of lactate removal (lactate oxidation complex) and also signal many of the adaptations in muscle found in response to endurance training. Although still controversial, the evidence provided in this and other recent publications is supportive of the ILS hypothesis, as well as for an adaptive role of muscle lactate production during exercise.
This work was supported by NIH R01 AR050459 to G.A.B. T.H. was supported by a grant from the Japan Society for the Promotion of Science. Thanks are due to Tamara Mau for reading and commenting on the manuscript.
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