Monocarboxylate Transporters Mct Slc16A

Many endogenous and exogenous short chain anionic compounds, such as lactic acid, pyruvate, acetoacetate, p-hydroxybutyrate, acetate, propionate, and butyrate, are all good substrates for a family of proton-coupled transporter proteins, termed the monocarboxylate transporter family (MCT; SLC16) (Price et al., 1998). Although investigation of MCT activity was studied biochemically in earlier literature, it was not until the first identified member of the MCT family, MCT1, was cloned in CHO cells (Kim et al., 1992), and later functionally expressed in a breast tumor cell line (Garcia et al., 1994), that the molecular characterization of this transporter family began (Enerson and Drewes, 2003). To date 14 members of the MCT family have been identified, each having unique tissue distribution (Halestrap and Price, 1999; Halestrap and Meredith, 2004). Given the relative size of this transporter family, this assessment will comprise only those MCT members relevant to the intestinal transport of pharmaceutical or therapeutic compounds. Additionally, given that the molecular and functional characterizations of members in this transporter family are still in their infancy, one is expected to consult the literature for a more comprehensive review (Price et al., 1998; Enerson and Drewes, 2003; Halestrap and Meredith, 2004). Finally, given the convoluted nomenclature for this transporter family, representative transporters will be identified by their MCT name, as opposed to SLC or another designation. Molecular and Structural Characteristics of Monocarboxylate Transporters

Kyte-Doolittle hydropathy plots indicate that MCT isoforms exhibit 12 putative TMD, with both the amine and carboxy termini located intracellularly (Price et al., 1998; Halestrap and Price, 1999). This has been confirmed experimentally for MCT1 in rat erythrocytes (Poole et al., 1996). Transporter topology varies with respect to the length of the carboxy-terminus after transmembrane domain 12 and a large intracellular loop located between TMD six and seven (Price et al., 1998); however, these structures are consistent among the MCT isoforms. MCT family members also exhibit two highly conserved motifs: [D/E]G[G/S][W/F][G/A]W which traverses into transmembrane domain one and YfXK[R/K][R/L]XLAX[G/A]XAXAG leading into transmembrane domain five, where residues shown in bold are conserved in all of the sequences, residues in square brackets indicate alternative amino acids, residues that are in normal type are the consensus amino acid at that position, and "X" represents any amino acid (Halestrap and Price, 1999). Site directed mutagenesis studies have been conducted for a number of residues, which have resulted in various changes in substrate specificities; however, most interesting is that mutagenesis of the highly conserved Arg313 in MCT1 results in reduction in affinity for lactate (Rahman et al., 1999). It has been surmised that this positively charged Arg binds the car-boxy anion of monocarboxylates, much like lactate dehydrogenase, allowing for transport (Poole and Halestrap, 1993; Carpenter and Halestrap, 1994; Halestrap and Price, 1999).

Seemingly another defining factor of MCT family members is the presence of ancillary proteins that are required for proper intracellular trafficking, cell surface expression, and/or function. Studies indicate MCT1, MCT3, and MCT4 require the presence of CD147 [also known as OX-47, extracellular matrix metal-loproteinase inducer (EMM-PRIN), HT7 or basigin], or the related protein GP70 (Embigen), both widely distributed cell surface glycoproteins, for proper cell surface expression and function (Philp et al., 2003). These glycoproteins exhibit a single transmembrane domain, two immunoglobulin-like domains in the extracellular region, and a short carboxy terminus cytoplasmic tail (Schuster et al., 1996). In contrast, MCT2 does not interact with CD147 specifically; however, it does appear to require an additional protein for proper expression at the cell surface (Kirk et al., 2000). The associations of MCT1 and MCT4 with CD147 have been confirmed by carboxy terminus tagging with fluorescent proteins (Kirk et al., 2000; Zhao et al., 2001). Lack of CD147 coexpression with MCT1 and MCT4 results in accumulation in the endoplasmic reticulum, or golgi apparatus (Kirk et al., 2000). Studies exploring the coexpression of CD147 or GP70 with other MCT isoforms have not been conducted. The Substrate Specificity of Monocarboxylate Transporters

Functional evidence for the proton-dependent symport of monocarboxylates via various MCT isoforms is well established (Enerson and Drewes, 2003).

Comparatively, MCT1, MCT2, and MCT4 exhibit higher affinities for pyruvate than lactate, although MCT4 exhibits low affinity for both substrates (Enerson and Drewes, 2003). The difference in affinities from pyruvate to lactate indicates a preference for 2-oxoacids over 2-hydroxy acids (Enerson and Drewes, 2003). Interestingly, the affinity for MCT2 for pyruvate is extremely high compared to MCT1 (Lin et al., 1998). Other monocarboxylates known to be substrates for MCTs include butyrate, acetate, propionate, etc. With respect to therapeutic compounds, it has been surmised that MCT isoforms may play a role in the intestinal absorption of some (3-lactam antibiotics (Li et al., 1999), penicillins (Itoh et al., 1998), nonsteroidal anti-inflammatory drugs (Emoto et al., 2002), valproic acid (Utoguchi and Audus, 2000; Hosoya et al., 2001), atorvastatin (Wu et al., 2000c), and nateglinide (Okamura et al., 2002).

One isoform in the MCT family, MCT10 (TAT1; SLC16A10), is an aromatic amino acid transporter that exhibits proton independent transport of substrates, although it does exhibit 30% identity to other MCTs (Kim et al., 2001a). The high sequence conservation (49%) between MCT10 and MCT8 suggests MCT8 may not function as a monocarboxylate transporter either (Friesema et al., 2005). Indeed, when expressed in Xenopus oocytes, MCT8 transports thyroid hormones T3 and T4 in a sodium and proton independent manner (Enerson and Drewes, 2003). Neither lactate nor aromatic amino acids appear to be substrates for MCT8 (Enerson and Drewes, 2003). Although the substrate affinities of MCT5-8 have not yet been identified, their similarity to MCT10 may also suggest an alternate substrate affinity, differing from MCT1-4 (Enerson and Drewes, 2003). The Expression of Monocarboxylate Transporters

Although each MCT isoform exhibits its own unique tissue distribution, of the 14 different MCTs currently identified, MCTs 1, 3-7 were shown to be expressed in the GI tract by Northern blotting (Price et al., 1998). However, at the time of that publication only seven MCT isoforms had been established and the intestinal expression of the remaining MCT isoforms has not yet been investigated. These northern blotting experiments also indicate that MCT2 is not intestinally localized, which has been supported by a recent publication exploring the intestinal distribution of a number of MCT isoforms (Gill et al., 2005). This literature suggests that MCT1, 4, and 5 are the predominant intestinally expressed isoforms, although MCT3 does exhibit some minimal expression (Gill et al., 2005). This study further suggests that MCT1 expression increases along the length of the human intestine, with a predominant expression in the distal colon, followed by proximal colon, ileum, and jejunum (Gill et al., 2005). Results were similar for MCT4 and MCT5, where expression increased moving down the GI tract; however, neither MCT4 nor MCT5 exhibited expression in human ileum (Gill et al., 2005). In contrast to previous data suggesting MCT3 is only expressed in the retinal epithelium (Yoon et al., 1997; Philp et al., 1998) when antibody specific to MCT3 was used at a very low dilution of 1:50, expression was observed in human GI tract. However, in contrast to MCT1, MCT4, and MCT5, expression was higher in ileum as compared to colonic regions (Gill et al., 2005). Expression of MCT6 was not observed in the human GI tract (Gill et al., 2005). Gill et al. (2005) also showed differences in the preferential membrane localization of these transporters, suggesting basolateral localization of MCT3, MCT4, and MCT5, while MCT1 is suggested as being apically localized (Gill et al., 2005). The baso-lateral expression of MCT10 has also been shown in human intestinal epithelial cells (Kim et al., 2001a,b). The Regulation of Monocarboxylate Transporters

With the exception of some limited studies conducted using MCT1, regulation mechanisms of the various intestinally expressed MCT isoforms has not been reported. Previous studies have shown that MCT1 is upregulated in response to excess butyrate substrate, which is reflected functionally as an increase in butyrate transport in cultured colonic epithelial cells (AA/C1) (Cuff et al., 2002). Moreover, it has been reported that MCT1 expression is decreased markedly during colon carcinogenesis, indicating a role of MCT1 in cancer prevention via active butyrate influx (Lambert et al., 2002). It has been proposed that the decrease in MCT1 expression during colon carcinogenesis may decrease the intracellular availability of butyrate required to regulate expression of genes associated with the processes maintaining tissue homeostasis within the colonic mucosa (Cuff et al., 2005). In fact, studies suggest that the ability of butyrate to induce cell-cycle arrest and differentiation is dependent on the abundance and functionality of MCT1 (Cuff et al., 2005). Furthermore, downregulation of MCT1 using RNAi in various colonic cell lines resulted in consistent inhibition of butyrate influx thus inhibiting its ability to modulate various indicators of carcinogenesis, namely IAP, a marker of differentiation, p21, a cell cycle inhibitor, and CD1, a positive regulator of cell cycle progression (Cuff et al., 2005). This study also concluded that MCT1 inhibition did not affect those genes associated with apoptosis, bcl-xL and bak(Cuff etal., 2005).

Whereas it is known that alteration in physiological state changes MCT expression, the underlying cellular and molecular mechanisms are poorly understood (Enerson and Drewes, 2003). However, studies of MCT regulation in other tissue do suggest an upregulation of MCT expression under hypoxia, as well as in the presence of exogenous vascular endothelial growth factor, which is known to be mediated by the hypoxia inducible factor HIF-1 (Enerson and Drewes, 2003). Moreover, mRNA expression of MCT1 in cultured macrophages is upregulated by exposure to lipopolysaccharides, tumor necrosis factor-a, and nitric oxide; however, only lipopolysaccharide and tumor necrosis factor-a treatment related to an increase in MCT1 protein (Hahn et al., 2000). These results suggest the presence of multiple signaling pathways that may converge to regulate MCT1 expression (Enerson and Drewes, 2003). Additionally, Leino et al. (2001) recently demonstrated upregulation of MCT1 and GLUT1 in rat brain after diet-induced ketosis. Results indicated an eightfold increase in MCT1 expression in brain endothelial cells after 4 weeks under ketonemic conditions (Leino et al., 2001). Moreover, an increase in MCT1 levels were shown throughout the rat brain, especially in the cerebellum, indicating protein upregulation in response to dietary and possibly pathological stresses (Leino et al., 2001).

Although the transport of monocarboxylates by MCTs is important with respect to several disease pathologies, such as cancer and ischemic stroke, given their widespread tissue expression (heart, brain, intestine, liver, etc.) and their substrate's impact on cellular energy and overall tissue and organism function, it is not expected that pharmaceutical intervention via MCTs is likely in the near future. This is not particularly surprising given the incomplete functional characterization and even identification of these transporters. However, the relative importance of MCTs to proper physiological function necessitates further research into the pathophysiology associated with this transporter family, especially considering the recent finding of the role of MCT8 dysfunction in causing severe X-linked psychomotor retardation, termed Allan-Herndon-Dudley syndrome (Dumitrescu et al., 2004; Friesema et al., 2004).

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