Shotaro Sasaki

Functional Characterization of 5-Oxoproline Transport via SLC16A1/MCT1

Background: The amino acid derivative 5-oxoproline, which is an endogenous compound in the brain, is a monocarboxylate. Results: Na+-dependent and amino acid transport systems scarcely contributed to 5-oxoproline transport in T98G cells as an astrocyte cell model. Conclusion: 5-Oxoproline is taken up only by the monocarboxylate transporter SLC16A1. Significance: 5-Oxoproline transport may be an important physiological function for SLC16A1. Thyrotropin-releasing hormone is a tripeptide that consists of 5-oxoproline, histidine, and proline. The peptide is rapidly metabolized by various enzymes. 5-Oxoproline is produced by enzymatic hydrolysis in a variety of peptides. Previous studies showed that 5-oxoproline could become a possible biomarker for autism spectrum disorders. Here we demonstrate the involvement of SLC16A1 in the transport of 5-oxoproline. An SLC16A1 polymorphism (rs1049434) was recently identified. However, there is no information about the effect of the polymorphism on SLC16A1 function. In this study, the polymorphism caused an observable change in 5-oxoproline and lactate transport via SLC16A1. The Michaelis constant (Km) was increased in an SLC16A1 mutant compared with that in the wild type. In addition, the proton concentration required to produce half-maximal activation of transport activity (K0.5, H+) was increased in the SLC16A1 mutant compared with that in the wild type. Furthermore, we examined the transport of 5-oxoproline in T98G cells as an astrocyte cell model. Despite the fact that 5-oxoproline is an amino acid derivative, Na+-dependent and amino acid transport systems scarcely contributed to 5-oxoproline transport. Based on our findings, we conclude that H+-coupled 5-oxoproline transport is mediated solely by SLC16A1 in the cells.

Crucial Residue Involved in L-Lactate Recognition by Human Monocarboxylate Transporter 4 (hMCT4)

Background Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4. Experimental approach Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4. Results In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization. Conclusions Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.

Involvement of Histidine Residue His382 in pH Regulation of MCT4 Activity

Monocarboxylate transporter 4 (MCT4) is a pH-dependent bi-directional lactate transporter. Transport of lactate via MCT4 is increased by extracellular acidification. We investigated the critical histidine residue involved in pH regulation of MCT4 function. Transport of lactate via MCT4 was measured by using a Xenopus laevis oocyte expression system. MCT4-mediated lactate transport was inhibited by Zn2+ in a pH physiological condition but not in an acidic condition. The histidine modifier DEPC (diethyl pyrocarbonate) reduced MCT4 activity but did not completely inactivate MCT4. After treatment with DEPC, pH regulation of MCT4 function was completely knocked out. Inhibitory effects of DEPC were reversed by hydroxylamine and suppressed in the presence of excess lactate and Zn2+. Therefore, we performed an experiment in which the extracellular histidine residue was replaced with alanine. Consequently, the pH regulation of MCT4-H382A function was also knocked out. Our findings demonstrate that the histidine residue His382 in the extracellular loop of the transporter is essential for pH regulation of MCT4-mediated substrate transport activity.

Regulation of multidrug resistance protein 2 (MRP2, ABCC2) expression by statins: Involvement of SREBP-mediated gene regulation

Multidrug resistance protein 2 (MRP2, ABCC2) is localized to the apical membrane of hepatocytes and played an important role in the biliary excretion of a broad range of endogenous and xenobiotic compounds and drugs, such as pravastatin. However, the effects of statins on MRP2 in the liver and the precise mechanisms of their actions have been obscure. The goal of this study was to determine the regulatory molecular mechanism for statin-induced MRP2 expression in hepatocytes. In vitro and in vivo studies suggested that pitavastatin increased MRP2 expression. Pitavastatin promoted liver X receptor (LXR) α/β translocation from the cytosol to nuclei, resulting in LXR activation. Deletion and mutational analysis suggested that the potential sterol regulatory element (SRE) played a major role in the observed modulation of MRP2 expression by pitavastatin. Furthermore pitavastatin increased the protein-DNA complex, and when SRE was mutated, stimulation of the protein-DNA complex by pitavastatin was decreased. It was demonstrated that pitavastatin upregulated MRP2 expression by an SREBP regulatory pathway in hepatocytes and that the actions of statins may lead to improve the biliary excretion of MRP2 substrates.

Influence of high glucose state on bromopyruvate-induced cytotoxity by human colon cancer cell lines.

BACKGROUND Attention must be paid to chemotherapy for cancer patients in a hyperglycemia state. It is difficult for chemotherapy to cure cancer in patients in a hyperglycemia state. This study was carried out to determine the change in cell viability after treatment with bromopyruvate, which is an alkylating drug with anti-tumor activity, in a high glucose condition. METHODS The function of l-lactate and bromopyruvate transport was studied using human colon cancer cell lines (LoVo and HT-29) and radiolabeled l-lactate and bromopyruvate. Cell viability was monitored by the trypan blue exclusion assay. The expression level of human monocarboxylate transporter 1 (hMCT1) was evaluated by Western blot analysis. RESULTS Bromopyruvate-induced cell death was suppressed by a high glucose condition. l-Lactate and bromopyruvate uptake were suppressed by a high glucose condition. hMCT1 as a bromopyruvate carrier was functionally expressed in the cells. However, the expression of hMCT1 was suppressed by a high glucose state. CONCLUSIONS Down-regulation of hMCT1 by a high glucose state is one of the possibilities of the bromopyruvate resistance. We should pay scrupulous attention to cancer chemotherapy for patients who have developed diabetes.

The flexible cytoplasmic loop 3 contributes to the substrate affinity of human monocarboxylate transporters

Human monocarboxylate transporters (hMCTs/SLC16As) mediate the transport of small molecular weight monocarboxylates. Among hMCTs, hMCT1 exhibits high-affinity l-lactate transport and broad substrate recognition, whereas hMCT4 shows highly specific substrate recognition and low-affinity l-lactate transport, indicating that hMCT1 and hMCT4 have different roles in the body. However, the molecular mechanism of transporter-mediated substrate transport remains unknown. The aim of this study is to identify the domain, which determines the substrate selectivity and affinity of hMCT1 and hMCT4. We constructed a chimera, hMCT4/1, in which the cytoplasmic loop 3 (TM6/7loop) region of hMCT4 was replaced by the corresponding region of hMCT1. Xenopus laevis oocyte heterologous expression system was used to characterize functional features of the chimera. We have demonstrated that the substrate affinity of hMCT1 and hMCT4 depends on the TM6/7loop. Non-conserved His237 residue in the TM6/7loop functions as a regulatory moiety of the substrate affinity. In contrast, the substrate selectivity of the transporters did not depend on the TM6/7loop, suggesting that the domain is not directly involved in substrate recognition. Our study provides important insights into the structures and functions of hMCT1 and hMCT4 transporters. These findings contribute to the development of novel hMCT1 and/or hMCT4 inhibitors as anticancer agents.

Interaction of atorvastatin with the human glial transporter SLC16A1

Solute carrier (SLC) 16A1 is a pH-dependent carrier of 5-oxoproline, a derivative of the amino acid. SLC16A1 interacts with carboxylate group-containing substrates, which are also present in atorvastatin, and might be the reason for its ability to interact with atorvastatin. Does atorvastatin interact with the carrier? Does it also interact with the carrier via the substrate recognition site? This study was carried out to answer these questions. Polymerase chain reaction was used to determine the expression of SLC16A1 in normal human astrocytes. We induced SLC16A1 expression in a mammalian cell line and in Xenopus laevis oocytes. We used [(3)H] 5-oxoproline for direct measurement of SLC16A1-specific transport activity. SLC16A1 was clearly observed in normal human astrocytes. 3-Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors inhibited the SLC16A1-specific transport of 5-oxoproline. Atorvastatin was the most potent inhibitor, with an inhibition constant of 40μM. The drug was a non-competitive inhibitor of SLC16A1. In the present study, we showed non-competitive inhibition of SLC16A1-specific transport activity by atorvastatin. However, the affinity between the drug and the carrier was extremely low. Therefore, the interaction of atorvastatin with SLC16A1 is unlikely to be a problem in clinical practice.

Involvement of monocarboxylate transporter 1 (SLC16A1) in the uptake of l-lactate in human astrocytes

Purpose: Astrocytes, the most abundant glial cells in the central nervous system (CNS), help neurons survive. Monocarboxylate transporters (MCTs) are reported to transport L‐lactate, which is important for CNS physiology and cognitive function. However, it remains unclear which MCT isoform is functionally expressed by human astrocytes. The aim of this study was to establish the contribution of each MCT isoform to L‐lactate transport in human astrocytes. Methods: The function of L‐lactate transport was studied using NHA cells as a human astrocyte model and radiolabeled L‐lactate. The expression of MCT in human astrocytes was detected by immunohistochemistry staining. Results: The cellular uptake of L‐lactate was found to be pH‐ and concentration‐dependent with a Km value for L‐lactate uptake of 0.64 mM. This Km was similar to what has been previously established for MCT1‐mediated L‐lactate uptake. &agr;‐Cyano‐4‐ hydroxycinnamate (CHC) and 5‐oxoproline, which are both MCT1 inhibitors, were found to significantly inhibit the uptake of L‐lactate, suggesting MCT1 is primarily responsible for L‐lactate transport. Moreover, MCT1 protein was expressed in human astrocytes. Conclusion: pH‐dependent L‐lactate transport is mediated by MCT1 in human astrocytes. Graphical abstract: Figure. No caption available. Highlights:Proton activation of L‐lactate transport in human astrocytes exhibits non‐linear kinetics with a Hill coefficient of 0.9.Uptake of L‐lactate in human astrocytes is concentration‐dependent with a Km value for L‐lactate uptake of 0.64 mM.MCT1 inhibitors significantly inhibit L‐lactate uptake in human astrocytes.