The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40 -60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2-3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.Lactic acid is both a major fuel for skeletal muscle ("red" oxidative fibers) and a major metabolic end product ("white" glycolytic muscles). Even oxidative fibers become net lactic acid exporters when oxygen supply cannot meet demand, and glycolysis is stimulated to maintain ATP supplies. Fatigue occurs when lactic acid builds up within the myocyte. This causes intracellular pH (pH i ) to drop, inhibiting both glycolysis and contractile activity (1, 2). In the extreme case further muscle activity is totally prevented, a phenomenon used to advantage by anglers "playing" their fish to exhaustion. The transport of lactic acid out of skeletal muscle fibers is essential if such intracellular accumulation of lactic acid is to be prevented.Better removal of lactic acid from the muscle fibers might improve athletic performance during intense exercise and enable better muscle function and subsequent recovery under pathological conditions such as inherited mitochondrial diseases, hypoxia, and reperfusion following a period of ischemia.Transport of lactic acid into skeletal muscle fibers for oxidation is thought to be mediated by the proton-linked monocarboxylate transporter (MCT) 1 isoform MCT1 whose expression correlates with the oxidative capacity of muscle fibers and is increased following chronic muscle stimulation (3, 4). However, sarcolemmal membranes of muscle fibers that are primarily glycolytic do not contain significant amounts of MCT1 yet transport lactic acid by means of a saturable carrier that is inhibited by known inhibitors of MCT1 (3,5,6). These data imply the presence of another MCT isoform in such glycolytic fibers. MCT kinetics in heart (7-9) and liver (10) cells also imply the existence of other MCT isoforms, and this conclusion has been confirmed by cloning and sequencing studies.The first MCT isoform (MCT1) was cloned from Chinese hamster ovary cells (11) and has since been cloned and sequenced from huma...

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The cDNA for the monocarboxylate transporter MCT2 from rat testis has been cloned and sequenced. The derived protein sequence shows 82% identity with that from hamster. Rat MCT2 has a relative insertion of five amino acids in the N-terminal sequence preceding the first predicted transmembrane segment. MCT2 appears to be less highly conserved between species than MCT1. Using Northern blotting of RNA from rat and mouse tissues, MCT2 message was demonstrated to be abundant in the testis where a smaller, less abundant MCT2 transcript was also present. Low levels of a slightly different-sized transcript were found in rat and mouse liver, and mouse kidney. In hamster, only one-size transcript was detected at relatively high abundance in all the tissues examined. Antibodies were raised against a peptide derived from the extreme C-terminus of rat MCT2, and Western blotting with these detected MCT2 in membrane fractions prepared from rat testis, liver and brain but not those from heart or skeletal muscle. In hamster, MCT2 was detected in liver, heart and testis but not in brain [Garcia, Brown, Pathak, and Goldstein (1995) J. Biol. Chem. 270, 1843-1849]. For both rat MCT1 and MCT2 there were marked differences between the relative abundance of their respective messages and the amount of protein in membrane fractions from different tissues. This suggests that expression of both of these transporters in different tissues may be species-specific and regulated post-transcriptionally. The different-sized MCT2 transcripts may arise from alternative splicing. Starvation of rats for up to 48 h did not lead to any change in MCT1 or MCT2 expression in the liver, as determined by either Northern or Western blotting.

The kinetics of transport of L-lactate, pyruvate, ketone bodies, and other monocarboxylates into isolated hepatocytes from starved rats were measured at 25°C using the intracellular pH-sensitive dye, 2 ,7 -bis(carboxyethyl)-5(6)-carboxyfluorescein, to detect the associated proton influx. Transport kinetics were similar, but not identical, to those determined using the same technique for the monocarboxylate transporter (

The extreme amino terminus and, in particular, residue Glu-3 in rat liver (L) carnitine palmitoyltransferase I (CPT I) have previously been shown to be essential for the sensitivity of the enzyme to inhibition by malonylCoA. Using the Pichia pastoris expression system, we now observe that, although mutants E3A (Glu-3 3 Ala) or ⌬(3-18) of L-CPT I have markedly lowered sensitivity to malonyl-CoA compared with the wild-type protein, the mutant ⌬(1-82) generated an enzyme that had regained much of the sensitivity of wild-type CPT I. This suggests that a region antagonistic to malonyl-CoA sensitivity is present within residues 19 -82 of the enzyme. This was confirmed in the construct ⌬(19 -30), which was found to be 50-fold more sensitive than wild-type L-CPT I. Indeed, this mutant was >4-fold more sensitive than even the native muscle (M)-CPT I isoform expressed and assayed under identical conditions. This behavior was dependent on the presence of Glu-3, with the mutant E3A-⌬(19 -30) having kinetic characteristics similar to those of the E3A mutant. The increase in the sensitivity of the L-CPT I-⌬(19 -30) mutant was not due to a change in the mechanism of inhibition with respect to palmitoyl-CoA, nor to any marked change of the K 0.5 for this substrate. Conversely, for M-CPT I, a decrease in malonyl-CoA sensitivity was invariably observed with increasing deletions from ⌬(3-18) to ⌬(1-80). However, deletion of residues 3-18 from M-CPT I affected the K m for carnitine of this isoform, but not of L-CPT I. These observations (i) provide the first evidence for negative determinants of malonyl-CoA sensitivity within the amino-terminal segment of L-CPT I and (ii) suggest a mechanism for the inverse relationship between affinity for malonyl-CoA and for carnitine of the two isoforms of the enzyme.Carnitine palmitoyltransferase I (CPT I, 1 malonyl-CoAsensitive) is an integral membrane protein first identified in the outer membrane and contact sites of mitochondria (1, 2). The enzyme catalyzes the formation of acylcarnitines from long-chain acyl-CoA esters, thus enabling the movement of acyl moieties across intracellular membranes. CPT I exists in two isoforms (Liver and Muscle), which have considerable sequence similarity but differ greatly, and inversely, in their malonyl-CoA sensitivity and K m for carnitine (3, 4). It is a polytopic protein, with two transmembrane (TM) segments and amino and carboxyl segments (approximately 46 and 652 residues, respectively) that are both exposed on the cytosolic aspect of the membrane (5). It has been shown that the extreme amino terminus of the nascent L-CPT I is retained in the mature protein (6) and, moreover, that it is essential for the expression of malonyl-CoA sensitivity (5, 7). Subsequent work with expressed CPT I constructs has confirmed these conclusions by showing that deletion of the amino-terminal highly conserved 6 amino acid residues of the L-isoform results in the loss of high affinity malonyl-CoA sensitivity (8). Glu-3, and to a much lesser extent His-5, have been identi...