CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.     

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.     

Co-expression of monocarboxylate transporter 1 (MCT1) and its chaperone (CD147) is associated with low survival in patients with gastrointestinal stromal tumors (GISTs)

Monocarboxylate transporters (MCTs) have been described to play an important role in cancer, but to date there are no reports on the significance of MCT expression in gastrointestinal stromal tumors (GISTs). The aim of the present work was to assess the value of MCT expression, as well as co-expression with the MCT chaperone CD147 in GISTs and evaluate their clinical-pathological significance. We analyzed the immunohistochemical expression of MCT1, MCT2, MCT4 and CD147 in a series of 64 GISTs molecularly characterized for KIT, PDGFRA and BRAF mutations. MCT1, MCT2 and MCT4 were highly expressed in GISTs. CD147 expression was associated with mutated KIT (p?=?0.039), as well as a progressive increase in Fletcher's Risk of Malignancy (p?=?0.020). Importantly, co-expression of MCT1 with CD147 was associated with low patient's overall survival (p?=?0.037). These findings suggest that co-expression of MCT1 with its chaperone CD147 is involved in GISTs aggressiveness, pointing to a contribution of cancer cell metabolic adaptations in GIST development and/or progression.     

Ruminal microbial digestion in free-living, in captive lichen-fed, and in starved reindeer (Rangifer tarandus tarandus) in winter.

In free-living (FL) reindeer eating a natural mixed winter diet dominated by lichens, captive (CF) reindeer fed pure lichens ad libitum, and CF reindeer subsequently starved for 1 day (CS1 reindeer) or 4 days (CS4 reindeer), the dominant rumen anaerobic bacteria were characterized, their population densities were estimated, and ruminal pH and volatile fatty acid concentrations were determined. In the FL reindeer, the total median viable anaerobic bacterial population ranged from 18 x 10(8) to 35 x 10(8) cells per ml of rumen fluid (n = 4), compared with 26 x 10(8) to 34 x 10(8) and 0.09 x 10(8) to 0.1 x 10(8) cells per ml of rumen fluid in CF reindeer (n = 2) and CS4 reindeer (n = 2), respectively. The median bacterial population adhering to the rumen solids ranged from 260 x 10(8) to 450 x 10(8), 21 x 10(8) to 38 x 10(8), and 0.5 x 10(8) cells per g (wet weight) of rumen solids in FL, CF, and CS4 reindeer, respectively. Although there were variations in the rumen bacterial composition among the FL reindeer (n = 4), strains of Bacteroides, Fibrobacter, Streptococcus, and Clostridium dominated in the rumen fluid. Streptococcus spp. and Clostridium spp. were the dominant bacteria in the CF reindeer (n = 2), while in the CS4 reindeer (n = 2) the dominant bacteria were Fusobacterium spp., members of the family Enterobacteriaceae, and Eubacterium spp. Transmission electron micrographs of lichen particles from the rumen of one FL reindeer, one CF reindeer, and one CS4 reindeer show bacteria resembling Bacteroides spp. adhering to the lichen particles, evidently digesting the lichen hyphae from the inside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subacute ruminal acidosis suppressed the expression of MCT1 in rumen of cows

Subacute ruminal acidosis (SARA) is characterized by the depression of ruminal pH and an increase in the concentrations of short-chain fatty acids (SCFAs) and lipopolysaccharide (LPS) in the rumen of cows. The onset of SARA was linked to the accumulation of SCFAs. However, the mechanism of SCFAs transport is unknown. The proton-linked monocarboxylate transporter (MCT1) plays a vital role in the transportation of SCFAs. The goal of this study was to elucidate the distribution of MCT1 along the gastrointestinal tract of calves and adult cows; the expression change of MCT1 in SARA cows and the effect of ruminal pH, SCFAs, and LPS on MCT1 expression in rumen epithelial cells in vitro. The results indicated the presence of MCT1 along the gastrointestinal tract of calves and adult cows, most abundantly expressed in the rumen. Importantly, the expression of MCT1 was decreased in the rumen epithelium of SARA cows, and the expression of MCT1 was restored in the SARA treatment group. In vitro, LPS, low rumen fluid pH, high concentrations of SCFAs (90 mM acetate, 40 mM propionate, and 30 mM butyrate), and high concentrations of acetate, propionate, and butyrate, respectively, inhibited the expression of MCT1 in rumen epithelial cells. Taken together, these results indicated that LPS, low ruminal pH, and high concentrations of SCFAs decreased the expression of MCT1, further aggravating the accumulation of SCFAs in the rumen by decreasing the absorption of SCFAs.     

Expression and membrane localization of MCT isoforms along the length of the human intestine

Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells. monocarboxylate transporter; short-chain fatty acids; absorption; short-chain fatty acid transport; mammalian colon     

GLUT1 and CAIX expression profiles in breast cancer correlate with adverse prognostic factors and MCT1 overexpression

The goal of the present work was to evaluate the correlation of glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX) with the monocarboxylate transporters 1 (MCT1) and 4 (MCT4) and their chaperone, CD147, in breast cancer. The clinico-pathological value of GLUT1 and CAIX was also evaluated. For that, we analysed the immunohistochemical expression of GLUT1 and CAIX, in a large series of invasive breast carcinoma samples (n=124), previously characterized for MCT1, MCT4 and CD147 expression. GLUT1 expression was found in 46% of the cases (57/124), while CAIX was found in 18% of the cases (22/122). Importantly, both MCT1 and CD147, but not MCT4, were associated with GLUT1 and CAIX expression. Also, GLUT1 and CAIX correlated with each other. Concerning the clinico-pathological values, GLUT1 was associated with high grade tumours, basal-like subtype, absence of progesterone receptor, presence of vimentin and high proliferative index as measured by Ki-67. Additionally, CAIX was associated with large tumour size, high histological grade, basal-like subtype, absence of estrogen and progesterone receptors and presence of basal cytokeratins and vimentin expression. Finally, patients with CAIX positive tumours had a significantly shorter disease-free survival. The association between MCT1 and both GLUT1 and CAIX may result from hypoxia-mediated metabolic adaptations, which confer a glycolytic, acid-resistant and more aggressive phenotype to cancer cells.     

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.     

Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4: the ancillary protein for the insensitive MCT2 is EMBIGIN (gp70)

Translocation of monocarboxylate transporters MCT1 and MCT4 to the plasma membrane requires CD147 (basigin) with which they remain tightly associated. However, the importance of CD147 for MCT activity is unclear. MCT1 and MCT4 are both inhibited by the cell-impermeant organomercurial reagent p-chloromercuribenzene sulfonate (pCMBS). Here we demonstrate by site-directed mutagenesis that removal of all accessible cysteine residues on MCT4 does not prevent this inhibition. pCMBS treatment of cells abolished co-immunoprecipitation of MCT1 and MCT4 with CD147 and enhanced labeling of CD147 with a biotinylated-thiol reagent. This suggested that CD147 might be the target of pCMBS, and further evidence for this was obtained by treatment of cells with the bifunctional organomercurial reagent fluorescein dimercury acetate that caused oligomerization of CD147. Site-directed mutagenesis of CD147 implicated the disulfide bridge in the Ig-like C2 domain of CD147 as the target of pCMBS attack. MCT2, which is pCMBS-insensitive, was found to co-immunoprecipitate with gp70 rather than CD147. The interaction between gp70 and MCT2 was confirmed using fluorescence resonance energy transfer between the cyan fluorescent protein- and yellow fluorescent protein-tagged MCT2 and gp70. pCMBS strongly inhibited lactate transport into rabbit erythrocytes, where MCT1 interacts with CD147, but not into rat erythrocytes where it interacts with gp70. These data imply that inhibition of MCT1 and MCT4 activity by pCMBS is mediated through its binding to CD147, whereas MCT2, which associates with gp70, is insensitive to pCMBS. We conclude that ancillary proteins are required to maintain the catalytic activity of MCTs as well as for their translocation to the plasma membrane.     

Bacteria in the small intestine of lichen-fed Norwegian reindeer (Rangifer tarandus tarandus)

It has been suggested that lactic acid-producing bacteria may protect the epithelium of the mammalian gastrointestinal tract from pathogenic micro-organisms. Consistent with this, bacteria isolated from the mucosa of the small intestine of five lichenfed, semi-domesticated reindeer included mainly Streptococcus spp. However, the population densities of bacteria associated with the mucosa and in the intestinal contents were generally low and there was a large amount of variation both between animals and with site of sampling. It therefore seems unlikely that Streptococcus spp. are essential for the function of the small intestine in captive reindeer, and their role here remains uncertain.     

Fluorescence resonance energy transfer studies on the interaction between the lactate transporter MCT1 and CD147 provide information on the topology and stoichiometry of the complex in situ.

The monocarboxylate (lactate) transporters MCT1 and MCT4 require the membrane-spanning glycoprotein CD147 for their correct plasma membrane expression and function. We have successfully expressed CD147 and MCT1 tagged on their C or N termini with either the cyan (CFP) or yellow (YFP) variants of green fluorescent protein. The tagged proteins were correctly targeted to the plasma membrane of COS-7 cells and were functionally active. Measurements of fluorescence resonance energy transfer (FRET) between all combinations of the tagged proteins were made. FRET was observed when either the C or N terminus of MCT1 (intracellular) is tagged with CFP or YFP and co-expressed with CD147 tagged with YFP or CFP on the C terminus (intracellular) but not the N terminus (extracellular). FRET was also observed between two CD147 molecules when both YFP and CFP were on the C terminus but not when both were on the N terminus or one on either end. No FRET was observed between MCT1-YFP and MCT-CFP in any combination. A wide range of controls including photobleaching were employed to confirm that where FRET was observed, it was not an artifact of direct excitation of YFP by the CFP excitation laser. It was also shown that nonspecific overcrowding of proteins did not induce FRET. Because FRET only occurs between two fluorophores if they are less than 100 A apart and in a suitable orientation, our data provide important information on the topology of CD147 and MCT1 within the plasma membrane. The minimum configuration consistent with the data is a dimer of CD147 associating with two MCT1 molecules such that the C terminus of CD147 in the cytosol is close to the C terminus of its partner CD147 and to the C and N termini of an associated MCT1 molecule. FRET may provide a non-invasive technique for measuring changes in these interactions in living cells.     

Monocarboxylic acid transporters, MCT1 and MCT2, in cortical astrocytes in vitro and in vivo

We used sequence-specific antibodies tocharacterize two monocarboxylic acid transporters, MCT1 and MCT2, inastrocytes. Both proteins are expressed in primary cultures of corticalastrocytes, as indicated by immunoblotting and immunofluorescence. BothMCT1 and MCT2 are present in small, punctate structures in thecytoplasm and at the cell membrane. Cells showing very low levels oflabeling for glial fibrillary acidic protein (GFAP) also label moredimly for MCT2, but not for MCT1. In vivo, double-labelimmunofluorescence studies coupled with confocal microscopy indicatethat MCT1 and MCT2 are rare in astrocytes in the cortex. However, theyare specifically labeled in astrocytes of the glial limiting membraneand in white matter tracts. Both transporters are also present in themicrovasculature. Comparison of labeling for MCT1 and MCT2 with markersof the blood-brain barrier shows that the transporters are not alwayslimited to the astrocytic endfeet in vivo. Our results suggestthat the level of expression of monocarboxylic acid transporters MCT1and MCT2 by cortical astrocytes in vivo is significantly lowerthan in vitro but that astrocytes in some other regions of thebrain can express one or both proteins in significant amounts.

     

Co-expression of a mammalian accessory trafficking protein enables functional expression of the rat MCT1 monocarboxylate transporter in Saccharomyces cerevisiae

We have developed a new heterologous expression system for monocarboxylate transporters. The system is based on a Saccharomyces cerevisiae pyk1 mae1 jen1 triple-deletion strain that is auxotrophic for pyruvate and deficient in monocarboxylate uptake. Growth of the yeast cells on ethanol medium supplemented with pyruvate or lactate was dependent on the expression of a suitable monocarboxylate transporter. We have used the system to characterize the functional significance of interactions between the rat MCT1 transporter and its ancillary protein CD147. CD147 was shown to improve trafficking of MCT1 to the plasma membrane and its uptake activity. Our results demonstrate a new strategy for the production of properly folded and correctly targeted membrane proteins in a microbial expression system by co-expression of appropriate accessory proteins.     

Effects of acute and chronic exercise on sarcolemmal MCT1 and MCT4 contents in human skeletal muscles: current status

Two lactate/proton cotransporter isoforms (monocarboxylate transporters, MCT1 and MCT4) are present in the plasma (sarcolemmal) membranes of skeletal muscle. Both isoforms are symports and are involved in both muscle pH and lactate regulation. Accordingly, sarcolemmal MCT isoform expression may play an important role in exercise performance. Acute exercise alters human MCT content, within the first 24 h from the onset of exercise. The regulation of MCT protein expression is complex after acute exercise, since there is not a simple concordance between changes in mRNA abundance and protein levels. In general, exercise produces greater increases in MCT1 than in MCT4 content. Chronic exercise also affects MCT1 and MCT4 content, regardless of the initial fitness of subjects. On the basis of cross-sectional studies, intensity would appear to be the most important factor regulating exercise-induced changes in MCT content. Regulation of skeletal muscle MCT1 and MCT4 content by a variety of stimuli inducing an elevation of lactate level (exercise, hypoxia, nutrition, metabolic perturbations) has been demonstrated. Dissociation between the regulation of MCT content and lactate transport activity has been reported in a number of studies, and changes in MCT content are more common in response to contractile activity, whereas changes in lactate transport capacity typically occur in response to changes in metabolic pathways. Muscle MCT expression is involved in, but is not the sole determinant of, muscle H(+) and lactate anion exchange during physical activity.     

Exercise-induced changes of MCT1 in cardiac and skeletal muscles of diabetic rats induced by high-fat diet and STZ

We hypothesized that a part of therapeutic effects of endurance training on insulin resistance is mediated by increase in cardiac and skeletal muscle mitochondrial lactate transporter, monocarboxylate transporter 1 (MCT1). Therefore, we examined the effect of 7 weeks endurance training on the mRNA and protein expression of MCT1 and MCT4 and their chaperon, CD147, on both sarcolemmal and mitochondrial membrane, separately, in healthy and type 2 diabetic rats. Diabetes was induced by injection of low dose of streptozotocin and feeding with high-fat diet. Insulin resistance was confirmed by homeostasis model assessment-estimated insulin resistance index and accuracy of two membranes separation was confirmed by negative control markers (glucose transporter 1 and cytochrome c oxidase. Real-time PCR and western blotting were used for mRNA and protein expression, respectively. Diabetes dramatically reduced MCT1 and MCT4 mRNA and their expression on sarcolemmal membrane whereas the reduction in MCT1 expression was less in mitochondrial membrane. Training increased the MCT1 mRNA and protein expression in both membranes and decreased insulin resistance as an adaptive consequence. In both tissues increase in CD147 mRNA was only parallel to MCT1 expression. The response of MCT1 on sarcolemmal and mitochondrial membranes was different between cardiac and skeletal muscles which indicate that intracellular lactate kinetic is tissue specific that allows a tissue to coordinate whole organism metabolism.     

Changes in MCT 1, MCT 4, and LDH expression are tissue specific in rats after long-term hypobaric hypoxia.

Little is known about the effect of chronic hypobaric hypoxia on the enzymes and transporters involved in lactate metabolism. We looked at the protein expression of monocarboxylate transporters MCT 1, MCT 2, and MCT 4, along with total lactate dehydrogenase (LDH) and LDH isozymes in skeletal muscle, cardiac muscle, and liver. Expression of these components of the lactate shuttle affects the ability to transport and oxidize lactate. We hypothesized that the expression of MCTs and LDH would increase after acclimation to high altitude (HA). The response to acclimation to HA was, however, tissue specific. In addition, the response was different in whole muscle (Mu) and mitochondria-enriched (Mi) fractions. Heart, soleus, and plantaris muscles showed the greatest response to HA. Acclimation resulted in a 34% increase in MCT 4 in heart and a decrease in MCT 1 (-47%) and MCT 4 (-47%) in plantaris Mu. In Mi fractions, the heart had an increase (+40%) and soleus a decrease (-40%) in LDH. HA also had a significant effect on the LDH isozyme composition of both the Mu and Mi fractions. Mitochondrial density was decreased in both the soleus (-17%) and plantaris (-44%) as a result of chronic hypoxia. We conclude that chronic hypoxia had a tissue-specific effect on MCTs and LDH (that form the lactate shuttle) but did not produce a consistent increase in these components in all tissues.     

Endocrinology of pregnancy and early pregnancy detection by reproductive hormones in reindeer (Rangifer tarandus tarandus)

The endocrinology was studied throughout pregnancy in reindeer (Rangifer tarandus tarandus) located in Oulu, Finland (65 degrees N, 25 degrees E) with 13 captive, semi domestic adult females. Blood samples were analyzed for plasma progesterone (P4), estradiol (E2) and estrone sulphate (E1SO4), 15-ketodihydro-PGF2alpha (PG-metabolite) and pregnancy associated glycoproteins (PAG). The mean plasma P4 concentration peaked twice during gestation: at around 24 and three weeks prior to calving. In pregnant females the plasma PAG concentration increased over basal concentrations 21-30 days after the estimated day of conception and peaked at the time of calving. The concentrations of E2 and E1SO4 remained low until 60 days before calving when a rapid increase was found for both hormones. The mean plasma concentration of PG-metabolite increased throughout pregnancy to a maximum at parturition. The estimated mean (range) gestation length was 216 (212-220) days. Judged from measures on reproductive organs collected from 86 free-ranging, semi-domestic female reindeer of unknown age presented for slaughter at Roros, Norway (63 degrees N, 11 degrees E) in the second week of December 1999, it was concluded that the breeding season lasted from early September until the end of November. The results also showed that plasma PAG concentration could provide a tool for detection of pregnancy in reindeer.     

Endurance training, expression, and physiology of LDH, MCT1, and MCT4 in human skeletal muscle

To evaluate the effects of endurance training on the expression of monocarboxylate transporters (MCT) in human vastus lateralis muscle, we compared the amounts of MCT1 and MCT4 in total muscle preparations (MU) and sarcolemma-enriched (SL) and mitochondria-enriched (MI) fractions before and after training. To determine if changes in muscle lactate release and oxidation were associated with training-induced changes in MCT expression, we correlated band densities in Western blots to lactate kinetics determined in vivo. Nine weeks of leg cycle endurance training [75% peak oxygen consumption (VO(2 peak))] increased muscle citrate synthase activity (+75%, P < 0.05) and percentage of type I myosin heavy chain (+50%, P < 0.05); percentage of MU lactate dehydrogenase-5 (M4) isozyme decreased (-12%, P < 0.05). MCT1 was detected in SL and MI fractions, and MCT4 was localized to the SL. Muscle MCT1 contents were consistent among subjects both before and after training; in contrast, MCT4 contents showed large interindividual variations. MCT1 amounts significantly increased in MU, SL, and MI after training (+90%, +60%, and +78%, respectively), whereas SL but not MU MCT4 content increased after training (+47%, P < 0.05). Mitochondrial MCT1 content was negatively correlated to net leg lactate release at rest (r = -0.85, P < 0.02). Sarcolemmal MCT1 and MCT4 contents correlated positively to net leg lactate release at 5 min of exercise at 65% VO(2 peak) (r = 0.76, P < 0.03 and r = 0. 86, P < 0.01, respectively). Results support the conclusions that 1) endurance training increases expression of MCT1 in muscle because of insertion of MCT1 into both sarcolemmal and mitochondrial membranes, 2) training has variable effects on sarcolemmal MCT4, and 3) both MCT1 and MCT4 participate in the cell-cell lactate shuttle, whereas MCT1 facilitates operation of the intracellular lactate shuttle.