Monocarboxylate transporters: past, present, and future.

We review here the 14 members of the Monocarboxylate transporter family (MCTs), their relationship based on sequence homology. The range of substrates transported by different members of this family extends from the standard monocarboxylate metabolites, lactic and pyruvic acids, to aromatic amino acids and thyroid hormones. The family is denoted Solute Carrier Family 16, or SLC16, among 43 SLC families constituting more than 300 members, which are annotated regularly at the website http://www.bioparadigms.org/slc/intro.htm. MCTs classically transport metabolites across plasma membranes with direction controlled by proton and metabolite concentrations independently of energy input, but they may also function in subcellular membranes. Their regulation may be complex, and they are implicated in leukocyte-mediated immunity, hypoxia induced cellular responses, and partitioning of the energy supply in several tissues. We focus here on histologic evidence (involving human tissue where available) and the first four 'classical' members; but we do annotate all 14, and note several candidate or proven genetic diseases that have arisen from MCT mutations. The review progresses through the following sections: (1) MCT1-4: genetics, kinetics, and modulation; (2) Chaperonins and targeting cofactors; (3) Tissue distribution of MCTs; (4) Intercellular lactate/pyruvate shuttles; (5) Transcriptional and translational regulation of MCTs; (6) Properties of other MCTs; and (7) Subcellular localization of MCTs and some future considerations. Along the way we posit questions or suggestions for future research.

Distribution of the monocarboxylate transporter MCT2 in human cerebral cortex: an immunohistochemical study.

The monocarboxylate transporter MCT2 belongs to a large family of membrane proteins involved in the transport of lactate, pyruvate and ketone bodies. Although its expression in rodent brain has been well documented, the presence of MCT2 in the human brain has been questioned on the basis of low mRNA abundance. In this study, the distribution of the monocarboxylate transporter MCT2 has been investigated in the cortex of normal adult human brain using an immunohistochemical approach. Widespread neuropil staining in all cortical layers was observed by light microscopy. Such a distribution was very similar in three different cortical areas investigated. At the cellular level, the expression of MCT2 could be observed in a large number of neurons, in fibers both in grey and white matter, as well as in some astrocytes, mostly localized in layer I and in the white matter. Double staining experiments combined with confocal microscopy confirmed the neuronal expression but also suggested a preferential postsynaptic localization of synaptic MCT2 expression. A few astrocytes in the grey matter appeared to exhibit MCT2 labelling but at low levels. Electron microscopy revealed strong MCT2 expression at asymmetric synapses in the postsynaptic density and also within the spine head but not in the presynaptic terminal. These data not only demonstrate neuronal MCT2 expression in human, but since a portion of it exhibits a distinct synaptic localization, it further supports a putative role for MCT2 in adjustment of energy supply to levels of activity.

Expression of the monocarboxylate transporter MCT1 in the adult human brain cortex.

Distribution of the monocarboxylate transporter MCT1 has been investigated in the cortex of normal adult human brain. Similarly to the glucose transporter GLUT1 55 kDa isoform, MCT1 was found to be strongly expressed on blood vessels in all cortical layers. In addition, laminar analysis revealed intense MCT1 expression in the neuropil of layer IV in primary auditory (AI) and visual (VI) areas, while this expression was more homogeneous in the non-primary auditory area STA. The cellular distribution shows that MCT1 is strongly expressed by glial cells often associated with blood vessels that were identified as astrocytes. The observed distribution of MCT1 supports the concept that, under certain circumstances, monocarboxylates could be provided as energy substrates to the adult human brain. Moreover, the distinct laminar pattern of MCT1 expression between primary and non-primary cortical areas may reflect different types of neuronal activity requiring adequate supply of specific energy substrates.

Expression of monocarboxylate transporter 4 in human platelets, leukocytes, and tissues assessed by antibodies raised against terminal versus pre-terminal peptides.

We compared antibodies (Abs) raised in rabbits against two non-overlapping peptides, terminal (T) and pre-terminal (PT) of the human monocarboxylate transporter (MCT4) lactate transporter in a variety of human tissues. Upon stringent SDS extraction, the PT Ab recognized a major 32 kDa band in many tissues, but not in leukocytes, while the T Ab recognized a 45 kDa band in leukocytes but only in a few other tissues. In two cell lines, human adult retinal pigment epithelial and Madin-Darby canine kidney, however, both Abs identified the same 45 kDa band only, whether extracted by stringent SDS or by a mild Triton X-100 procedure. Applying Triton X-100 and milder SDS methods to human tissues led us to conclude that: (1) MCT4 is more labile to proteolysis than MCT1 or 2; (2) the proteolysis involves an enzyme system which is absent from the cell lines, is of variable content in human tissues, and is accelerated by SDS and/or heat; (3) a major product is the 32 kDa band, which is missing the C-terminal peptide, since it is seen by the Ab to MCT4-PT, but not the Ab to MCT4-T; (4) this truncated 32 kDa form is prone to aggregate, producing oligomers also detected only by the MCT4-PT; (5) the 32 kDa form may have a physiological function, since (except in the cell lines and monocytes) it is the major form seen with the PT Ab even with our mildest extractions, and since MCT4-PT stained two compartments that were not stained by the T Ab in our immunohistochemistry survey: the capsule of the muscle spindle, and the cytoplasm of the lymphocyte; (6) platelets contained MCT4, stained by both Abs, and verified by the 45kDa band on Western blotting, in addition to the presence of MCT2 that we had demonstrated previously [N. Merezhinskaya, S.A. Ogunwuyi, F.G. Mullick, W.N. Fishbein, Presence and localization of three lactic acid transporters (MCT1, -2, and -4) in separated human granulocytes, lymphocytes, and monocytes, J. Histochem. Cytochem. 52 (2004) 1483-1493.

Presence and localization of three lactic acid transporters (MCT1, -2, and -4) in separated human granulocytes, lymphocytes, and monocytes.

We fractionated leukocytes from three donors into >90% pure samples of granulocytes, lymphocytes, and monocytes and tested them for transcriptional and translational expression of three physiologically-proven lactate transporters, monocarboxylate transporter 1(MCT1), MCT2, and MCT4, using RT-PCR and affinity-purified rabbit antibody (Ab) to the C-terminal segment of each human MCT. Transcripts of all three MCTs were identified in each leukocyte fraction by RT-PCR and proven by sequencing of fragments extracted after isolation on agarose gels. Transporter protein of the appropriate size was demonstrated for each of the monocarboxylate transporters MCTs in lymphocytes and monocytes by Western blot, while lower-molecular-weight bands were found in granulocytes and are presumed to be degraded forms, because they were blocked by antibody-antigen (Ab-Ag) preincubation. IHC demonstrated all three MCTs in methanol-fixed droplets of all three leukocyte fractions; stain was abolished on omission of the primary Ab. Plasmalemmal staining occurred with all MCTs in all leukocyte fractions. Because the K(m) for lactate increases approximately fivefold at each step, with MCT2<1<4, leukocytes must use the full range of lactate binding to survive in acidic and hypoxic environments. Except for MCT4 in lymphocytes, all the MCTs also stained leukocyte cytoplasm, often with distinct granularity. Nuclear membrane staining was also seen with MCT1 and MCT2, while platelet plasmalemma stained only with MCT2.

Relative distribution of three major lactate transporters in frozen human tissues and their localization in unfixed skeletal muscle.

We have prepared affinity-purified rabbit polyclonal antibodies to the near-C-terminal peptides of human monocarboxylate transporters (MCTs) 1, 2, and 4 coupled to keyhole limpet hemocyanin. Each antiserum reacted only with its specific peptide antigen and gave a distinct molecular weight band (blocked by preincubation with antigen) after chemiluminescence reaction on Western blots from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of tissue membrane proteins. Densitometry showed distinctive expression patterns for each MCT in a panel of 15 frozen human tissues, with the distribution of MCT1 >>MCT2>MCT4. Fluorescence microscopy of unfixed skeletal muscle using fluorescein-conjugated secondary antibody was correlated with reverse adenosine triphosphatase (ATPase) stained sequential sections to identify fiber-type localization. MCT1 expression was high in the sarcolemma of type 1 fibers, modest to low in type 2a fibers, and almost absent in type 2b fibers. In contrast, MCT4 expression was low to absent in the membrane of most type 1 fibers, but high in most 2a and in all 2b fibers, favoring the view that their high lactate levels during work may be channeled in part to neighboring type 1 (and perhaps 2a) fibers for oxidation, thereby delaying fatigue. MCT2 expression was limited to the sarcolemma of a type 1 fiber subset, which varied from <5 to 40%, depending on the specific muscle under study. Quantitative chemiluminescent densitometry of 10 muscle biopsies for their MCT2 and MCT4 content, each normalized to MCT1, confirmed the unique variation of MCT2 expression with biopsy site. The application of these antibodies should add to the understanding of motor unit physiology, and may contribute to the muscle-biopsy assessment of low-level denervation.